Histochemical characteristics of the basement membranes of the parietal podocytes in the rabbit kidney. A light- and electron-microscopic study

The patterns of silver affinity and following treatment with guanidine were studied in the basement membrane produced by pareital podocytes induced by corticoids in newborn rabbits. The goal of this study was to analyze the role of the different cell types of the renal corpuscle in the determination of the histochemical characteristics of the glomerular basement membrane (GBM). Jones' method shows that while the GBM exhibited silver affinity only after periodic-acid oxidation, the basement membrane of parietal podocytes exhibits the same histochemical characteristics as the normal parietal basement membrane, appearing deep black both after periodic-acid or permanganate oxidation, and after elastase or lysozyme digestions. Since the treatment with guanidine shows that the basement membrane of the parietal podocytes lacks the endothelial component typical of the GBM, it may be suggested that the special resistance to silver impregnation exhibited by the basement membrane after permanganate oxidation or after different enzymatic digestions is due to its endothelial component.     

(Pro)renin receptor and autophagy in podocytes

Within the kidneys, podocytes are highly specialized postmitotic cells. Podocytes, together with endothelial cells and the glomerular basement membrane (GBM), maintain the filtration barrier and the normal structure of the glomerular capillary, are involved in the remodeling of the GBM and the endocytosis of filtered proteins, and counteract intracapillary hydrostatic pressure. The (pro)renin receptor [(P)RR], as ATP6AP2, is an accessory subunit of the vacuolar H⁺-ATPase, implying more fundamental developmental functions for the (pro)renin receptor in addition to its role in activating the local renin-angiotensin system, and is also expressed in podocytes, where it is involved in both tissue angiotensin II production and (P)RR-mediated intracellular signaling. Overexpression of human (P)RR in rats caused slowly progressive proteinuria and glomerular sclerosis, which suggests that (P)RR-mediated signaling is involved in the development of glomerular diseases. However, the physiological role of the (P)RR in podocytes has not yet been fully understood.     

(Pro)renin receptor and autophagy in podocytes

Within the kidneys, podocytes are highly specialized postmitotic cells. Podocytes, together with endothelial cells and the glomerular basement membrane (GBM), maintain the filtration barrier and the normal structure of the glomerular capillary, are involved in the remodeling of the GBM and the endocytosis of filtered proteins, and counteract intracapillary hydrostatic pressure. The (pro)renin receptor [(P)RR], as ATP6AP2, is an accessory subunit of the vacuolar H (+) -ATPase, implying more fundamental developmental functions for the (pro)renin receptor in addition to its role in activating the local renin-angiotensin system, and is also expressed in podocytes, where it is involved in both tissue angiotensin II production and (P)RR-mediated intracellular signaling. Overexpression of human (P)RR in rats caused slowly progressive proteinuria and glomerular sclerosis, which suggests that (P)RR-mediated signaling is involved in the development of glomerular diseases. However, the physiological role of the (P)RR in podocytes has not yet been fully understood.     

Mechanisms and consequences of TGF-? overexpression by podocytes in progressive podocyte disease

In patients with progressive podocyte disease, such as focal segmental glomerulosclerosis (FSGS) and membranous nephropathy, upregulation of transforming growth factor-? (TGF-?) is observed in podocytes. Mechanical pressure or biomechanical strain in podocytopathies may cause overexpression of TGF-? and angiotensin II (Ang II). Oxidative stress induced by Ang II may activate the latent TGF-?, which then activates Smads and Ras/extracellular signal-regulated kinase (ERK) signaling pathways in podocytes. Enhanced TGF-? activity in podocytes may lead to thickening of the glomerular basement membrane (GBM) by overproduction of GBM proteins and impaired GBM degradation in podocyte disease. It may also lead to podocyte apoptosis and detachment from the GBM, and epithelial-mesenchymal transition (EMT) of podocytes, initiating the development of glomerulosclerosis. Furthermore, activated TGF-?/Smad signaling by podocytes may induce connective tissue growth factor and vascular endothelial growth factor overexpression, which could act as a paracrine effector mechanism on mesangial cells to stimulate mesangial matrix synthesis. In proliferative podocytopathies, such as cellular or collapsing FSGS, TGF-?-induced ERK activation may play a role in podocyte proliferation, possibly via TGF-?-induced EMT of podocytes. Collectively, these data bring new mechanistic insights into our understanding of the TGF-? overexpression by podocytes in progressive podocyte disease.     

Pathogenic role of TGF-β in the progression of podocyte diseases

In patients with progressive podocyte diseases, such as focal segmental glomerulosclerosis and membranous nephropathy, there is enhanced expression of transforming growth factor (TGF-β) in podocytes. Biomechanical strain in these diseases may cause overexpression of TGF-β and angiotensin II (Ang II) by podocytes. Oxidative stress induced by Ang II may activate the latent TGF-β. Increased TGF-β activity by podocytes may induce not only the thickening of the glomerular basement membrane (GBM), but also podocyte apoptosis and/or detachment from the GBM, initiating the development of glomerulosclerosis. Furthermore, mesangial matrix expansion frequently occurs in podocyte diseases in association with the development of glomerulosclerosis. This review examines open questions on the pathogenic role of TGF-β that links podocyte injury to GBM thickening, podocyte loss, mesangial matrix expansion and glomerulosclerosis in podocyte diseases. It also describes paracrine regulatory mechanisms of podocyte TGF-β on mesangial cells leading to increased matrix synthesis.     

Altering expression of alpha3beta1 integrin on podocytes of human and rats with diabetes

The adhesion molecule integrin alpha3beta1 is the major receptor of podocyte to the glomerular capillary basement membrane (GBM). Since progressive alteration of the glomerular extracellular matrix (ECM) compartment leading to GBM thickening is common in diabetic nephropathy, we investigated the cellular distribution of alpha3beta1 integrin in podocytes of patients with diabetic nephropathy and streptozotocin-induced diabetic rats, and we evaluated the effects of high glucose on the cultured rat podocytes. Both human and rat kidneys were stained using the immunoelectron microscopy and immunoperoxidase technique with mouse monoclonal antibodies to human integrin alpha3 subunit. The results showed that both the number of immunogold particles and the staining of integrin alpha3 subunit on podocytes were weaker in patients with diabetic nephropathy than those of control kidneys. The staining of alpha3 on podocytes in the poorly-controlled diabetic rats was also weaker after one and three months of hyperglycemia. However, the staining was identical to controls in rats with only one week of hyperglycemia. High glucose (25 mM) but not streptozotocin in vitro suppressed the alpha3 expression of cultured rat podocytes. Our results demonstrated that the expression of integrin alpha3beta1 on podocytes was suppressed in both human and rats with diabetes, possibly due to the effects of hyperglycemia, and the suppression became more severe with the duration of diabetes.     

Freeze-fracture cytochemistry of rat glomerular capillary tuft. Determination of wheat germ agglutinin binding sites and localization of anionic charges

We propose here the use of freeze-fracture to gain access and to label in vitro glomerular components and locate WGA receptors and anionic sites. Tissues are frozen, fractured under liquid nitrogen, and thawed. Freeze-fracture rendered all glomerular structures directly accessible to the reagents. This made possible study of the nature and topology of cationized ferritin and WGA binding sites. WGA-gold complexes were observed over plasma membranes of podocytes and of endothelial and mesangial cells. Labeling of podocytes and endothelial cells was similar in the mesangial area and in the peripheral part of the capillary loop. Cross-fractures of extracellular matrices showed that WGA bound uniformly to the glomerular basement membrane (GBM) as well as to mesangial matrix. In fractured specimens treated with neuraminidase, WGA was no longer observed over podocytes but it consistently labeled the surface of endothelial and mesangial cells. Whereas in GBM cross-sections WGA binding was greatly reduced or even abolished, it remained unmodified in the mesangium. This shows that only NeuNAc (sialic acid) might account for the binding of WGA to podocytes, whereas GlcNAcs appear to be the main WGA binding sites on endothelial and mesangial cells and in the mesangial matrix. Both NeuNAc and GLcNAc residues are probably associated in GBM. With cationized ferritin (pI 8.3) at pH 7.4, intense, continuous labeling was seen all over the different plasma membranes, denser in podocytes than in endothelial cells. CF was also observed in cross-fractured profiles of extracellular matrices and never appeared agglutinated in discrete sites.     

Integrin beta1-mediated matrix assembly and signaling are critical for the normal development and function of the kidney glomerulus

The human kidneys filter 180 l of blood every day via about 2.5 million glomeruli. The three layers of the glomerular filtration apparatus consist of fenestrated endothelium, specialized extracellular matrix known as the glomerular basement membrane (GBM) and the podocyte foot processes with their modified adherens junctions known as the slit diaphragm (SD). In this study we explored the contribution of podocyte β1 integrin signaling for normal glomerular function. Mice with podocyte specific deletion of integrin β1 (podocin-Cre β1-fl/fl mice) are born normal but cannot complete postnatal renal development. They exhibit detectable proteinuria on day 1 and die within a week. The kidneys of podocin-Cre β1-fl/fl mice exhibit normal glomerular endothelium but show severe GBM defects with multilaminations and splitting including podocyte foot process effacement. The integrin linked kinase (ILK) is a downstream mediator of integrin β1 activity in epithelial cells. To further explore whether integrin β1-mediated signaling facilitates proper glomerular filtration, we generated mice deficient of ILK in the podocytes (podocin-Cre ILK-fl/fl mice). These mice develop normally but exhibit postnatal proteinuria at birth and die within 15 weeks of age due to renal failure. Collectively, our studies demonstrate that podocyte β1 integrin and ILK signaling is critical for postnatal development and function of the glomerular filtration apparatus.     

Cysteine cathepsins B,X and K expression in peri-arteriolar glioblastoma stem cell niches

Glioblastoma (GBM) is the most lethal brain tumor also due to malignant and therapy-resistant GBM stem cells (GSCs) that are localized in protecting hypoxic GSC niches. Some members of the cysteine cathepsin family of proteases have been found to be upregulated in GBM. Cathepsin K gene expression is highly elevated in GBM tissue versus normal brain and it has been suggested to regulate GSC migration out of the niches. Here, we investigated the cellular distribution of cathepsins B, X and K in GBM tissue and whether these cathepsins are co-localized in GSC niches. Therefore, we determined expression of these cathepsins in serial paraffin sections of 14 human GBM samples and serial cryostat sections of two samples using immunohistochemistry and metabolic mapping of cathepsin activity using selective fluorogenic substrates. We detected cathepsins B, X and K in peri-arteriolar GSC niches in 9 out of 16 GBM samples, which were defined by co-expression of the GSC marker CD133, the niche marker stromal-derived factor-1α (SDF-1α) and smooth muscle actin as a marker for arterioles. The expression of cathepsin B and X was detected in stromal cells and cancer cells throughout the GBM sections, whereas cathepsin K expression was more restricted to arteriole-rich regions in the GBM sections. Metabolic mapping showed that cathepsin B, but not cathepsin K is active in GSC niches. On the basis of these findings, it is concluded that cathepsins B, X and K have distinct functions in GBM and that cathepsin K is the most likely GSC niche-related cathepsin of the three cathepsins investigated.     

Evidence for splicing new basement membrane into old during glomerular development in newborn rat kidneys

Tannic acid in glutaraldehyde fixatives greatly enhanced the visualization of two developmentally and morphologically distinct stages in glomerular basement membrane (GBM) formation in newborn rat kidneys. First, in early stage glomeruli, double basement membranes between endothelial cells and podocytes were present and, in certain areas, appeared to be fusing. Second, in maturing stage glomeruli, elaborate loops and outpockets of basement membrane projected into epithelial, but not endothelial, sides of capillary walls. When Lowicryl thin sections from newborn rat kidneys were sequentially labeled with rabbit anti-laminin IgG and anti-rabbit IgG-colloidal gold, gold bound across the full width of all GBMs, including double basement membranes and outpockets. The same distribution was obtained when sections from rats that received intravenous injections of rabbit anti-laminin IgG 1 h before fixation were labeled directly with anti- rabbit IgG-colloidal gold. When kidneys were fixed 4 d after anti- laminin IgG injection, however, loops beneath the podocytes in maturing glomeruli were usually unlabeled and lengths of unlabeled GBM were interspersed with labeled lengths. In additional experiments, rabbit anti-laminin IgG was intravenously injected into newborn rats and, 4-14 d later, rats were re-injected with sheep anti-laminin IgG. Sections were then doubly labeled with anti-rabbit and anti-sheep IgG coupled to 10 and 5 nm colloidal gold, respectively. Sheep IgG occurred alone in outpockets of maturing glomeruli and also in lengths of GBM flanked by lengths containing rabbit IgG. These results indicate that, after fusion of double basement membranes, new segments of GBM appear beneath developing podocytes and are subsequently spliced into existing GBM. This splicing provides the additional GBM necessary for expanding glomerular capillaries.     

Mutation analysis of LMX1B gene in nail-patella syndrome patients.

Nail-patella syndrome (NPS), a pleiotropic disorder exhibiting autosomal dominant inheritance, has been studied for >100 years. Recent evidence shows that NPS is the result of mutations in the LIM-homeodomain gene LMX1B. To determine whether specific LMX1B mutations are associated with different aspects of the NPS phenotype, we screened a cohort of 41 NPS families for LMX1B mutations. A total of 25 mutations were identified in 37 families. The nature of the mutations supports the hypothesis that NPS is the result of haploinsufficiency for LMX1B. There was no evidence of correlation between aspects of the NPS phenotype and specific mutations.