Imaging the Kidney with an Unconventional Scanning Electron Microscopy Technique: Analysis of the Subpodocyte Space in Diabetic Mice.

Transmission electron microscopy (TEM) remains the gold standard for renal histopathological diagnoses, given its higher resolving power, compared with light microscopy. However, it imposes several limitations on pathologists, including longer sample preparation time and a small observation area. To overcome these, we introduced a scanning electron microscopy (SEM) technique for imaging resin-embedded semi-thin sections of renal tissue. We developed a rapid tissue preparation protocol for experimental models and human biopsies which, alongside SEM digital imaging acquisition of secondary electrons (SE-SEM), enables fast electron microscopy examination, with a resolution similar to that achieved by TEM. We used this unconventional SEM imaging approach to investigate the subpodocyte space (SPS) in BTBR ob/ob mice with type 2 diabetes. Analysis of semi-thin sections with secondary electrons revealed that the SPS had expanded in volume and covered large areas of the glomerular basement membrane, forming wide spaces between the podocyte body and the underlying filtering membrane. Our results show that SE-SEM is a valuable tool for imaging the kidney at the ultrastructural level, filling the magnification gap between light microscopy and TEM, and reveal that in diabetic mice, the SPS is larger than in normal controls, which is associated with podocyte damage and impaired kidney function.

Different sample preparation methods for glomerular morphological evaluation: to coat or not to coat? / Diferentes métodos de preparación de muestras para la evaluación morfológica glomerular ¿recubrir o no recubrir?

The renal glomerulus is coated by fenestrated endothelial cells and externally covered by specialized epithelial cells, known as podocytes. Scanning electron microscopy becomes an important and effective tool for its studies. Normally, samples destined for scanning microscopy are covered with a thin metallic layer. However, this step can be dispensed for some analyzes. We aimed to compare coated and uncoated samples for evaluation of the glomerular morphology of the Wistar rat kidney. Cortical region of the kidney of the 5month-old male Wistar rats were used. The fragments followed the routine procedure for scanning electron microscopy processing. Half of 10 fragments were coated with palladium gold and the remaining were not coated. Auriga Compact FIB - SEM scanning electron microscope was used to observe the samples. Different increases and voltages was evaluated. For the uncoated samples, when using voltages of 2 KV (or higher) a great charging was observed, impairing the use of such voltage. Thus, these samples were always observed under voltage of 0.5 KV. On the other hand, in the coated samples, the use of 2 KV was adequate. Almost as a consequence, in the coated samples, the podocyte structures were better characterized, generating better images. Inversely, in the uncoated samples, it was possible to visualize the desired structures and to detect the morphological characteristics of these. The results showed that it is possible to use kidney samples without previous coating to evaluate the glomerular morphology at the ultrastructural level, serving as a tool in the study of pathologies.

El glomérulo renal está recubierto por células endoteliales fenestradas y cubierto externamente por células epiteliales especializadas, conocidas como podocitos. La microscopía electrónica de barrido se convierte en una herramienta importante y efectiva para sus estudios. Normalmente, las muestras destinadas a microscopía de barrido se cubren con una capa metálica delgada. Sin embargo, este paso se puede dispensar para algunos análisis. El objetivo fue comparar muestras recubiertas y no recubiertas para evaluar la morfología glomerular del riñón de rata Wistar. Se utilizó la región cortical del riñón de ratas Wistar macho de 5 meses de edad. Se realizó el procedimiento de rutina para el procesamiento de microscopía electrónica de barrido. La mitad de 10 fragmentos se recubrieron con oro paladio y los restantes no se recubrieron. Se utilizó un microscopio electrónico de barrido SEM Auriga Compact FIB para observar las muestras. Se evaluaron diferentes aumentos y voltajes. Para las muestras no recubiertas, al usar voltajes de 2 KV (o más) se observó una gran carga, impidiendo el uso de dicho voltaje. Por lo tanto, estas muestras siempre se observaron a bajo voltaje de 0,5 KV. Por otro lado, en las muestras recubiertas, el uso de 2 KV fue adecuado. Como consecuencia, en las muestras recubiertas, las estructuras de los podocitos se caracterizaron mejor, generando mejores imágenes. Inversamente, en las muestras no recubiertas, fue posible visualizar las estructuras deseadas y detectar las características morfológicas de éstas. Los resultados mostraron que es posible utilizar muestras de riñón sin recubrimiento previo para evaluar la morfología glomerular a nivel ultraestructural, que sirve como una herramienta en el estudio de patologías.

Nephrotic syndrome in adults.

Nephrotic syndrome is an important presentation of glomerular disease characterised by heavy proteinuria, hypoalbuminaemia and oedema. The differential diagnosis of the underlying condition is wide including primary renal disorders and secondary diseases such as malignancy, infection, diabetes and amyloid. Presentations to acute medicine may be with hypervolaemia, complications of the nephrotic state (such as venous thromboembolism), or complications of therapy (such as infection). Early recognition of nephrotic syndrome is possible through simple urinalysis for protein and testing serum albumin, although a high index of suspicion is sometimes required in patients with comorbidities including potentially distracting cardiac or hepatic diseases.

Podocyte-actin dynamics in health and disease.

Genetic studies of hereditary forms of nephrotic syndrome have identified several proteins that are involved in regulating the permselective properties of the glomerular filtration system. Further extensive research has elucidated the complex molecular basis of the glomerular filtration barrier and clearly established the pivotal role of podocytes in the pathophysiology of glomerular diseases. Podocyte architecture is centred on focal adhesions and slit diaphragms - multiprotein signalling hubs that regulate cell morphology and function. A highly interconnected actin cytoskeleton enables podocytes to adapt in order to accommodate environmental changes and maintain an intact glomerular filtration barrier. Actin-based endocytosis has now emerged as a regulator of podocyte integrity, providing an impetus for understanding the precise mechanisms that underlie the steady-state control of focal adhesion and slit diaphragm components. This Review outlines the role of actin dynamics and endocytosis in podocyte biology, and discusses how molecular heterogeneity in glomerular disorders could be exploited to deliver more rational therapeutic interventions, paving the way for targeted medicine in nephrology.

Renal tubular damage may contribute more to acute hyperglycemia induced kidney injury in non-diabetic conscious rats.

AIMS: Growing evidences suggest that acute hyperglycemia is strongly related to kidney injury. Our study aimed to investigate the effects of acute hyperglycemia on kidney glomerular and tubular impairment in non-diabetic conscious rats. METHODS: Non-diabetic conscious rats were randomly subjected to 6h of saline (control group) or high glucose (acute hyperglycemia group) infusion. Blood glucose was maintained at 16.0-18.0 mmol/L in acute hyperglycemia group. Renal structure and function alterations, systemic/renal inflammation and oxidative stress markers were assessed, and apoptosis markers of renal inherent cells were evaluated. RESULTS: Acute hyperglycemia caused significant injury to structure of glomerular filtration barrier, tubular epithelial cells and peritubular vascular endothelial cells. It increased urinary microalbumin (68.01 ± 27.09 µg/24h vs 33.81 ± 13.81 µg/24h , P=0.014), ß2-microglobulin, Cystatin C, urinary and serous neutrophil gelatinase-associated lipocalin levels (P < 0.05). Acute hyperglycemia decreased megalin and cubilin expression, activated systemic and renal oxidative stress as well as inflammation and promoted renal inherent cell apoptosis. CONCLUSIONS: Acute hyperglycemia causes significant injury to kidney function and structure. Compared with damages of glomerular filtration barrier, renal tubular injury may contribute more to acute hyperglycemia induced proteinuria. Activation of inflammation especially renal inflammation, oxidative stress and enhanced apoptosis may be the underlying mechanisms.

Injury to the endothelial surface layer induces glomerular hyperfiltration rats with early-stage diabetes.

Glomerular endothelial surface layer (ESL) may play a role in the mechanisms of albuminuria in diabetic nephropathy, which lack evidence in vivo. The effects of high glucose on the passage of albumin across the glomerular ESL were analysed in streptozotocin-induced diabetic Sprague-Dawley rats for 4 weeks. Albuminuria and glomerular mesangial matrix were significantly increased in diabetic rats. The passage of albumin across the ESL, as measured by albumin-colloid gold particle density in the glomerular basement membrane (GBM), was increased significantly in diabetic rats. The thickness of the glomerular ESL, examined indirectly by infusing Intralipid into vessels using an electron microscope, was significantly decreased and the GBM exhibited little change in diabetic rats. In summary, the glomerular ESL may play a role in the pathogenesis of albuminuria in rats with early-stage diabetes.

Resolution of the three dimensional structure of components of the glomerular filtration barrier.

BACKGROUND: The human glomerulus is the primary filtration unit of the kidney, and contains the Glomerular Filtration Barrier (GFB). The GFB had been thought to comprise 3 layers - the endothelium, the basement membrane and the podocyte foot processes. However, recent studies have suggested that at least two additional layers contribute to the function of the GFB, the endothelial glycocalyx on the vascular side, and the sub-podocyte space on the urinary side. To investigate the structure of these additional layers is difficult as it requires three-dimensional reconstruction of delicate sub-microscopic (<1 µm) cellular and extracellular elements. METHODS: Here we have combined three different advanced electron microscopic techniques that cover multiple orders of magnitude of volume sampled, with a novel staining methodology (Lanthanum Dysprosium Glycosaminoglycan adhesion, or LaDy GAGa), to determine the structural basis of these two additional layers. Serial Block Face Scanning Electron Microscopy (SBF-SEM) was used to generate a 3-D image stack with a volume of a 5.3 x 105 µm3 volume of a whole kidney glomerulus (13% of glomerular volume). Secondly, Focused Ion Beam milling Scanning Electron Microscopy (FIB-SEM) was used to image a filtration region (48 µm3 volume). Lastly Transmission Electron Tomography (Tom-TEM) was performed on a 0.3 µm3 volume to identify the fine structure of the glycocalyx. RESULTS: Tom-TEM clearly showed 20 nm fibre spacing in the glycocalyx, within a limited field of view. FIB-SEM demonstrated, in a far greater field of view, how the glycocalyx structure related to fenestrations and the filtration slits, though without the resolution of TomTEM. SBF-SEM was able to determine the extent of the sub-podocyte space and glycocalyx coverage, without additional heavy metal staining. Neither SBF- nor FIB-SEM suffered the anisotropic shrinkage under the electron beam that is seen with Tom-TEM. CONCLUSIONS: These images demonstrate that the three dimensional structure of the GFB can be imaged, and investigated from the whole glomerulus to the fine structure of the glycocalyx using three dimensional electron microscopy techniques. This should allow the identification of structural features regulating physiology, and their disruption in pathological states, aiding the understanding of kidney disease.

Application of transmission electron tomography for modeling the renal corpuscle.

Structural alteration to the microanatomical organization of the glomerular filtration barrier results in proteinuria. Conventional transmission electron microscopy is an important diagnostic tool to assess the degree of ultrastructural damage of the corpusclar filtration unit. However, this approach lacks the ability to collect accurate stereological insights in a relative large tissue volume. Transmission electron tomography offers the ability to gather three-dimensional information with relative ease. Therefore, this contribution aims to highlight what electron tomography can bring to the pathologist in this challenging area of diagnostic practice. Kidney tissue was prepared for routine ultrastructural transmission electron microscopy investigation. Three-dimensional data stacks were automatically acquired by tilting semi-thin sections of 270 nm in an angular range of typically -60° to +60° with 1° increment. Subsequently, models of the filtration unit were produced by computer-assisted tracking of structures of interest. This short report illustrates the capability that transmission electron tomography can offer in the fine structure-function assessment of the porous fenestrated glomerular capillary endothelium, the underlying basement membrane and the podocyte filtration slits. Furthermore, this approach allows the generation of morphometric data about size, shape and volume alterations of the kidney's filtration barrier at the nanoscale.

Glomerular filtration barrier in pediatric idiopathic nephrotic syndrome.

Nephrotic syndrome (NS) is a common proteinuric disorder with defect in the perm-selectivity of the glomerular filtration barrier (GFB). Ultrastructural morphometric evaluation of the GFB in pediatric NS has been attempted in only a few studies. This study was aimed at qualitative and quantitative evaluation of the alterations involving the GFB in pediatric idiopathic NS with an attempt to correlate these alterations with the clinico-laboratory data. For this study, renal biopsies from nine patients with NS and two children with interstitial nephritis were included. Relevant clinical and laboratory data, including degree of 24-h proteinuria and renal function tests, were recorded. Renal biopsies were reviewed for morphologic and electron microscopic diagnosis. Ultrastructural morphometry of the GFB was performed using image analysis software. The age at onset of NS, duration of illness, presence of hypertension, and renal function tests were comparable between the group of patients with minimal change disease (MCD) and those with mesangioproliferative glomerulonephritis (mesPGN)/focal segmental glomerulosclerosis (FSGS). However, the latter group showed higher 24-h proteinuria compared with the group with MCD. Among the detected ultra-structural changes, glomerular basement membrane thickness and foot process width were significantly different between the MCD and the mesPGN/FSGS groups. The slit pore diameter in the glomeruli showed a positive correlation with the degree of proteinuria. We conclude that our study demonstrated remarkable differences in certain parameters and the glomerular ultrastructural alterations in the various categories of NS. These differences might underlie the observed variation in response of these entities to various therapies.

The adaptor protein Grb2 is not essential for the establishment of the glomerular filtration barrier.

The kidney filtration barrier is formed by the combination of endothelial cells, basement membrane and epithelial cells called podocytes. These specialized actin-rich cells form long and dynamic protrusions, the foot processes, which surround glomerular capillaries and are connected by specialized intercellular junctions, the slit diaphragms. Failure to maintain the filtration barrier leads to massive proteinuria and nephrosis. A number of proteins reside in the slit diaphragm, notably the transmembrane proteins Nephrin and Neph1, which are both able to act as tyrosine phosphorylated scaffolds that recruit cytoplasmic effectors to initiate downstream signaling. While association between tyrosine-phosphorylated Neph1 and the SH2/SH3 adaptor Grb2 was shown in vitro to be sufficient to induce actin polymerization, in vivo evidence supporting this finding is still lacking. To test this hypothesis, we generated two independent mouse lines bearing a podocyte-specific constitutive inactivation of the Grb2 locus. Surprisingly, we show that mice lacking Grb2 in podocytes display normal renal ultra-structure and function, thus demonstrating that Grb2 is not required for the establishment of the glomerular filtration barrier in vivo. Moreover, our data indicate that Grb2 is not required to restore podocyte function following kidney injury. Therefore, although in vitro experiments suggested that Grb2 is important for the regulation of actin dynamics, our data clearly shows that its function is not essential in podocytes in vivo, thus suggesting that Grb2 rather plays a secondary role in this process.

Transmembrane collagen XVII is a novel component of the glomerular filtration barrier.

The kidney filtration barrier consists of the capillary endothelium, the glomerular basement membrane and the slit diaphragm localized between foot processes of neighbouring podocytes. We report that collagen XVII, a transmembrane molecule known to be required for epithelial adhesion, is expressed in podocytes of normal human and mouse kidneys and in endothelial cells of the glomerular filtration barrier. Immunoelectron microscopy has revealed that collagen XVII is localized in foot processes of podocytes and in the glomerular basement membrane. Its role in kidney has been analysed in knockout mice, which survive to birth but have high neonatal mortality and skin blistering and structural abnormalities in their glomeruli. Morphometric analysis has shown increases in glomerular volume fraction and surface densities of knockout kidneys, indicating an increased glomerular amount in the cortex. Collagen XVII deficiency causes effacement of podocyte foot processes; however, major slit diaphragm disruptions have not been detected. The glomerular basement membrane is split in areas in which glomerular and endothelial basement membranes meet. Differences in the expression of collagen IV, integrins α3 or ß1, laminin α5 and nephrin have not been observed in mutant mice compared with controls. We propose that collagen XVII has a function in the attachment of podocyte foot processes to the glomerular basement membrane. It probably contributes to podocyte maturation and might have a role in glomerular filtration.

Sepsis induces albuminuria and alterations in the glomerular filtration barrier: a morphofunctional study in the rat.

INTRODUCTION: Increased vascular permeability represents one of the hallmarks of sepsis. In the kidney, vascular permeability is strictly regulated by the 'glomerular filtration barrier' (GFB), which is comprised of glomerular endothelium, podocytes, their interposed basement membranes and the associated glycocalyx. Although it is likely that the GFB and its glycocalyx are altered during sepsis, no study has specifically addressed this issue. The aim of this study was to evaluate whether albuminuria--the hallmark of GFB perm-selectivity--occurs in the initial stage of sepsis and whether it is associated with morphological and biochemical changes of the GFB. METHODS: Cecal ligation and puncture (CLP) was used to induce sepsis in the rat. Tumor necrosis factor (TNF)-alpha levels in plasma and growth of microorganisms in the peritoneal fluid were evaluated at 0, 3 and 7 hours after CLP or sham-operation. At the same times, kidney specimens were collected and structural and ultrastructural alterations in the GFB were assessed. In addition, several components of GFB-associated glycocalyx, syndecan-1, hyluronan (HA) and sialic acids were evaluated by immunofluorescence, immunohistochemistry and lectin histochemistry techniques. Serum creatinine and creatinine clearance were measured to assess kidney function and albuminuria for changes in GFB permeability. Analysis of variance followed by Tukey's multiple comparison test was used. RESULTS: Septic rats showed increased TNF-alpha levels and growth of microorganisms in the peritoneal fluid. Only a few renal corpuscles had major ultrastructural and structural alterations and no change in serum creatinine or creatinine clearance was observed. Contrarily, urinary albumin significantly increased after CLP and was associated with diffuse alteration in the glycocalyx of the GFB, which consisted in a decrease in syndecan-1 expression and in HA and sialic acids contents. Sialic acids were also changed in their structure, exhibiting a higher degree of acetylation. CONCLUSIONS: In its initial phase, sepsis is associated with a significant alteration in the composition of the GFB-associated glycocalyx, with loss of GFB perm-selectivity as documented by albumin leakage into urine.