Novel Microscopic Techniques for Podocyte Research

Together with endothelial cells and the glomerular basement membrane, podocytes form the size-specific filtration barrier of the glomerulus with their interdigitating foot processes. Since glomerulopathies are associated with so-called foot process effacement—a severe change of well-formed foot processes into flat and broadened processes—visualization of the three-dimensional podocyte morphology is a crucial part for diagnosis of nephrotic diseases. However, interdigitating podocyte foot processes are too narrow to be resolved by classic light microscopy due to Ernst Abbe's law making electron microscopy necessary. Although three dimensional electron microscopy approaches like serial block face and focused ion beam scanning electron microscopy and electron tomography allow volumetric reconstruction of podocytes, these techniques are very time-consuming and too specialized for routine use or screening purposes. During the last few years, different super-resolution microscopic techniques were developed to overcome the optical resolution limit enabling new insights into podocyte morphology. Super-resolution microscopy approaches like three dimensional structured illumination microscopy (3D-SIM), stimulated emission depletion microscopy (STED) and localization microscopy [stochastic optical reconstruction microscopy (STORM), photoactivated localization microscopy (PALM)] reach resolutions down to 80–20 nm and can be used to image and further quantify podocyte foot process morphology. Furthermore, in vivo imaging of podocytes is essential to study the behavior of these cells in situ. Therefore, multiphoton laser microscopy was a breakthrough for in vivo studies of podocytes in transgenic animal models like rodents and zebrafish larvae because it allows imaging structures up to several hundred micrometer in depth within the tissue. Additionally, along with multiphoton microscopy, lightsheet microscopy is currently used to visualize larger tissue volumes and therefore image complete glomeruli in their native tissue context. Alongside plain visualization of cellular structures, atomic force microscopy has been used to study the change of mechanical properties of podocytes in diseased states which has been shown to be a culprit in podocyte maintenance. This review discusses recent advances in the field of microscopic imaging and demonstrates their currently used and other possible applications for podocyte research

MDM2 prevents spontaneous tubular epithelial cell death and acute kidney injury

Murine double minute-2 (MDM2) is an E3-ubiquitin ligase and the main negative regulator of tumor suppressor gene p53. MDM2 has also a non-redundant function as a modulator of NF-kB signaling. As such it promotes proliferation and inflammation. MDM2 is highly expressed in the unchallenged tubular epithelial cells and we hypothesized that MDM2 is necessary for their survival and homeostasis. MDM2 knockdown by siRNA or by genetic depletion resulted in demise of tubular cells in vitro. This phenotype was completely rescued by concomitant knockdown of p53, thus suggesting p53 dependency. In vivo experiments in the zebrafish model demonstrated that the tubulus cells of the larvae undergo cell death after the knockdown of mdm2. Doxycycline-induced deletion of MDM2 in tubular cell-specific MDM2-knockout mice Pax8rtTa-cre; MDM2f/f caused acute kidney injury with increased plasma creatinine and blood urea nitrogen and sharp decline of glomerular filtration rate. Histological analysis showed massive swelling of renal tubular cells and later their loss and extensive tubular dilation, markedly in proximal tubules. Ultrastructural changes of tubular epithelial cells included swelling of the cytoplasm and mitochondria with the loss of cristae and their transformation in the vacuoles. The pathological phenotype of the tubular cell-specific MDM2-knockout mouse model was completely rescued by co-deletion of p53. Tubular epithelium compensates only partially for the cell loss caused by MDM2 depletion by proliferation of surviving tubular cells, with incomplete MDM2 deletion, but rather mesenchymal healing occurs. We conclude that MDM2 is a non-redundant survival factor for proximal tubular cells by protecting them from spontaneous p53 overexpression-related cell death

Comparative Analysis of Podocyte Foot Process Morphology in Three Species by 3D Super-Resolution Microscopy

Since the size selectivity of the filtration barrier and kidney function are highly dependent on podocyte foot process morphology, visualization of foot processes is important. However, the size of foot processes is below the optical resolution of light microscopy. Therefore, electron microcopy has been indispensable to detect changes in foot process morphology so far, but it is a sophisticated and time-consuming technique. Recently, our group has shown that 3D structured illumination microscopy (3D-SIM), a super-resolution microscopy (SRM) technique, can visualize individual foot processes in human biopsies. Moreover, we have developed a software-based approach to directly quantify the structure of podocyte foot processes named Podocyte Exact Morphology Measurement Procedure (PEMP). As shown in patients suffering from minimal change disease (MCD), PEMP allows the quantification of changes of the foot process morphology by measuring the filtration slit density (FSD). Since rodents are frequently used in basic research, we have applied PEMP to quantify foot processes of mice and rats. Comparative analysis of nephrin-stained kidneys from humans, rats, and mice showed significant differences of the FSD. The highest FSD was measured in mice (3.83 ± 0.37 μm−1; mean ± SD) followed by rats (3.36 ± 0.42 μm−1) and humans (3.11 ± 0.26 μm−1). To demonstrate that PEMP can be used to determine foot process morphology also in affected animals, we measured the FSD in palladin-knockout mice on a 129S1 genetic background compared to wild-type littermates. Taken together, we established a method for the quick and exact quantification of podocyte foot process morphology which can be applied to diagnosis and basic research

Arp3 controls the podocyte architecture at the kidney filtration barrier

Podocytes, highly specialized epithelial cells, build the outer part of the kidney filtration barrier and withstand high mechanical forces through a complex network of cellular protrusions. Here, we show that Arp2/3-dependent actin polymerization controls actomyosin contractility and focal adhesion maturation of podocyte protrusions and thereby regulates formation, maintenance, and capacity to adapt to mechanical requirements of the filtration barrier. We find that N-WASP-Arp2/3 define the development of complex arborized podocyte protrusions in vitro and in vivo. Loss of dendritic actin networks results in a pronounced activation of the actomyosin cytoskeleton and the generation of over-maturated but less efficient adhesion, leading to detachment of podocytes. Our data provide a model to explain podocyte protrusion morphology and their mechanical stability based on a tripartite relationship between actin polymerization, contractility, and adhesion

CTNSmRNA as a potential treatment for nephropathic cystinosis

Messenger RNA (mRNA) therapies are emerging in different disease areas, but have not yet reached the kidney field. Our aim was to study the feasibility to correct the genetic defect in nephropathic cystinosis using synthetic mRNA. Cystinosis is a prototype disorder of proximal tubular dysfunction caused by mutations in the CTNS gene, encoding the lysosomal cystine-H+ symporter cystinosin, and leading to cystine accumulation in all cells of the body. The kidneys are the first and most severely affected organs, presenting glomerular and proximal tubular dysfunction. Cysteamine is the current therapeutic standard that reduces cellular cystine levels, but has many side effects and does not restore kidney function. Here, we show that synthetic mRNA is safe and effective to reintroduce functional cystinosin using lipofection in CTNS-/- kidney cells and following direct injection in ctns-/- zebrafish larvae. CTNS mRNA therapy results in prompt lysosomal expression of the functional protein and decreases cellular cystine accumulation for up to 14 days. In the ctns-/- zebrafish, CTNS mRNA therapy improves proximal tubular reabsorption, reduces proteinuria, and restores brush border expression of the multi-ligand receptor megalin. We propose that mRNA-based therapy, if sufficient kidney targeting can be achieved, may be a new approach to treat cystinosis

BDNF: mRNA expression in urine cells of patients with chronic kidney disease and its role in kidney function

Podocyte loss and changes to the complex morphology are major causes of chronic kidney disease (CKD). As the incidence is continuously increasing over the last decades without sufficient treatment, it is important to find predicting biomarkers. Therefore, we measured urinary mRNA levels of podocyte genes NPHS1, NPHS2, PODXL and BDNF, KIM-1, CTSL by qRT-PCR of 120 CKD patients. We showed a strong correlation between BDNF and the kidney injury marker KIM-1, which were also correlated with NPHS1, suggesting podocytes as a contributing source. In human biopsies, BDNF was localized in the cell body and major processes of podocytes. In glomeruli of diabetic nephropathy patients, we found a strong BDNF signal in the remaining podocytes. An inhibition of the BDNF receptor TrkB resulted in enhanced podocyte dedifferentiation. The knockdown of the orthologue resulted in pericardial oedema formation and lowered viability of zebrafish larvae. We found an enlarged Bowman's space, dilated glomerular capillaries, podocyte loss and an impaired glomerular filtration. We demonstrated that BDNF is essential for glomerular development, morphology and function and the expression of BDNF and KIM-1 is highly correlated in urine cells of CKD patients. Therefore, BDNF mRNA in urine cells could serve as a potential CKD biomarker

Four-dimensional in vivo imaging of glomerular morphology and glomerular barrier function in a zebrafish podocyte injury model

The global prevalence of kidney diseases has been steadily rising over the last decades. Today, around 10% of the world population suffers from relevant chronic kidney disease. Podocytes are highly specialized and terminally differentiated cells residing in the filtering units of the kidneys, the so-called glomeruli. With their interdigitating foot-processes, these cells are a crucial part of the renal filtration barrier. As podocytes are post-mitotic, injury or loss of these cells results in an impairment of the filtration barrier with subsequent loss of global kidney function. Therefore, the question whether a relevant amount of podocytes can be regenerated and if this regeneration can be influenced is crucial for future therapeutic developments. As in vivo microscopic imaging of podocytes in higher animals like mice or rats is rather challenging, larval zebrafish have been applied as an animal model for podocyte development and kidney filtration. 48 hours post fertilization, zebrafish larvae develop a single filtering glomerulus with a similar morphology and molecular construction to that in mammals. For evaluation of podocyte morphology and filtration, we used transgenic zebrafish strains in which podocytes were labeled with fluorescence proteins. Additionally, podocytes expressed the bacterial enzyme nitroreductase fused to the fluorescence protein mCherry. In this model, application of the antibiotic metronidazole leads to podocyte-specific cell death. Through cross-breeding we established strains that additionally express an eGFP-labeled protein in the blood plasma. Using in vivo two-photon microscopy, we could image podocyte-loss induced impairments of the glomerular filtration barrier. Additionally, we tracked characteristic morphological changes of podocyte morphology including podocyte foot process effacement, development of sub-podocyte pseudocysts and finally detachment of whole cells from the glomerular basement membrane. These changes have been before described histologically in different animal models as well as in patient biopsies. Using the in vivo microscopy approach, we could clearly describe the temporal sequence of these alterations. Finally, we also tracked individual, non-detached podocytes over up to 24 hours, and found that these cells were non-migratory. These results show that early podocyte-regeneration through immigration of intra- or extraglomerular cells is unlikely within the first 24 hours of acute glomerular injury.In den letzten Jahren stieg die globale Prävalenz von Nierenerkrankungen stetig. Heute leiden rund 10% der Weltbevölkerung an relevanter chronischer Niereninsuffizienz. Podozyten sind hochspezialisierte, terminal differenzierte Zellen in den Filtereinheiten der Niere, den sogenannten Glomeruli. Mit ihren interdigitierenden Fußfortsätzen sind diese Zellen ein unverzichtbarer Teil der renalen Filtrationsbarriere. Da Podozyten postmitotisch sind, führen Schädigung oder Verlust dieser Zellen zum Zusammenbruch der selektiven Filtrationsfunktion der Niere mit folgendem globalem Verlust der Nierenfunktion. Daher ist die Frage, ob und wenn ja auf welchem Wege Podozyten regeneriert werden können relevant für künftige Therapieentwicklungen. Unter anderem da die in vivo Beobachtung von Podozyten in höheren Versuchsorganismen schwierig ist wird seit Jahren die Zebrafischlarve als beliebter Modellorganismus für die Podozytenentwicklung und Nierenfiltration genutzt. 48 Stunden nach der Befruchtung entwickeln Zebrafischlarven ein einzelnes filtrierendes Glomerulus mit, im Vergleich zu Säugern, konserviertem histologischen und molekularem Aufbau. Zur Evaluation von Podozytenmorphologie und –funktion nutzten wir transgene Stämme in denen Podozyten durch Fluoreszenzproteine markiert wurden und das Enzym Nitroreduktase exprimierten. Die Applikation des Antibiotikums Metronidazol führt dadurch spezifisch zum Zelltod von Podozyten. Durch Kreuzungen erzeugten wir Stämme, in denen zusätzlich ein eGFP-markiertes Fluoreszenzprotein im Blutplasma als Marker für die Integrität der Filtrationsbarriere exprimiert wurde. Durch in vivo Zwei-Photonenmikroskopie konnten wir dadurch den Verlust der glomerulären Filtrationsbarriere und die charakteristischen Änderungen der podozytären Morphologie in der Podozytenschädigung verfolgen. Diese Änderungen umfassten den Verlust der podozytären Verzweigungen, Entwicklung subpodozytärer Pseudozysten und Ablösung ganzer Zellen von der glomerulären Basalmembran. Diese Veränderungen wurden zuvor histologisch in verschiedenen Tiermodellen und auch Patientenbiopsien beschrieben. Durch den in vivo Mikroskopischen Ansatz konnten wir diese Veränderungen klar in eine zeitliche Abfolge bringen. Zusätzlich verfolgten wir einzelne, verbliebene Podozyten über bis zu 24 Stunden und fanden heraus, dass diese Zellen nicht mobil waren. Diese Ergebnisse machen eine frühe Podozytenregeneration durch Immigration extraglomerulärer Zellen innerhalb der ersten 24 Stunden unwahrscheinlich.

Super-resolution microscopy: A technique to revolutionize research and diagnosis of glomerulopathies

For decades, knowledge about glomerular (patho)physiology has been tightly linked with advances in microscopic imaging technology. For example, the invention of electron microscopy was required to hypothesize about the mode of glomerular filtration barrier function. Super-resolution techniques, defined as fluorescence microscopy approaches that surpass the optical resolution limit of around 200 nm, have been made available to the scientific community. Several of these different techniques currently are in use in glomerular research. Using three-dimensional structured illumination microscopy, the exact morphology of the podocyte filtration slit can be morphometrically analyzed and quantitatively compared across samples originating from animal models or human biopsies. Several quantitative image analysis approaches and their potential influence on glomerular research and diagnostics are discussed

The Impact of Lidocaine on Adipose-Derived Stem Cells in Human Adipose Tissue Harvested by Liposuction and Used for Lipotransfer

The local anesthetic lidocaine, which has been used extensively during liposuction, has been reported to have cytotoxic effects and therefore would be unsuitable for use in autologous lipotransfer. We evaluated the effect of lidocaine on the distribution, number, and viability of adipose-derived stem cells (ASCs), preadipocytes, mature adipocytes, and leukocytes in the fatty and fluid portion of the lipoaspirate using antibody staining and flow cytometry analyses. Adipose tissue was harvested from 11 female patients who underwent liposuction. Abdominal subcutaneous fat tissue was infiltrated with tumescent local anesthesia, containing lidocaine on the left and lacking lidocaine on the right side of the abdomen, and harvested subsequently. Lidocaine had no influence on the relative distribution, cell number, or viability of ASCs, preadipocytes, mature adipocytes, or leukocytes in the stromal-vascular fraction. Assessing the fatty and fluid portions of the lipoaspirate, the fatty portions contained significantly more ASCs (p < 0.05), stem cells expressing the preadipocyte marker Pref-1 (p < 0.01 w/lidocaine, p < 0.05 w/o lidocaine), and mature adipocytes (p < 0.05 w/lidocaine, p < 0.01 w/o lidocaine) than the fluid portions. Only the fatty portion should be used for transplantation. This study found no evidence that would contraindicate the use of lidocaine in lipotransfer. Limitations of the study include the small sample size and the inclusion of only female patients