Biology of glomerular cells in culture

The glomerulus is a complex structure including four cell types, namely mesangial, visceral epithelial, parietal epithelial and endothelial cells. Mesangial cells resemble smooth muscle cells and play a major role in the synthesis of the components of the glomerular basement membrane and in the vasoreactivity of the glomerular tuft. In particular, they express receptors for angiotensin II which mediate mesangial cell contraction, this effect resulting in the decrease of the filtration area. They are also the site of synthesis of a variety of inflammatory agents which are involved in the development of glomerular injury in glomerulonephritis. Visceral epithelial cells, also referred to a podocytes, also participate in the synthesis of the normal constituents of the glomerular basement membrane. They express receptors for atrial natriuretic factor and possess on their surface a number of ectoenzymes. They also, in concert with mesangial cells, release metalloproteases which contribute to the degradation of the extracellular matrix. Parietal epithelial cells have been little studied. They represent the main constituent of the crescents observed in extracapillary proliferative glomerulonephritis. Endothelial cells secrete vasodilatory agents such as nitric oxide and prostacyclin and vasoconstrictor agents such as endothelin which act on the adjacent mesangial cells. New methods of culture of glomerular cells are in progress. Their aim is to keep as long as possible the physiological phenotype of these cells. Another progress is the availability of stable transformed cell lines which represent an abundant source of material for biochemical studies.     

Role of the polarity protein Scribble for podocyte differentiation and maintenance

The kidney filter represents a unique assembly of podocyte epithelial cells that tightly enwrap the glomerular capillaries with their complex foot process network. While deficiency of the polarity proteins Crumbs and aPKC result in impaired podocyte foot process architecture, the function of basolateral polarity proteins for podocyte differentiation and maintenance remained unclear. Here we report, that Scribble is expressed in developing podocytes, where it translocates from the lateral aspects of immature podocytes to the basal cell membrane and foot processes of mature podocytes. Immunogold electron microscopy reveals membrane associated localisation of Scribble predominantly at the basolateral site of foot processes. To further study the role of Scribble for podocyte differentiation Scribble(flox/flox) mice were generated by introducing loxP-sites into the Scribble introns 1 and 8 and these mice were crossed to NPHS2.Cre mice and Cre deleter mice. Podocyte-specific Scribble knockout mice develop normally and display no histological, ultrastructural or clinical abnormalities up to 12 months of age. In addition, no increased susceptibility to glomerular stress could be detected in these mice. In contrast, constitutive Scribble knockout animals die during embryonic development indicating the fundamental importance of Scribble for embryogenesis. Like in podocyte-specific Scribble knockout mice, the development of podocyte foot processes and the slit diaphragm was unaffected in kidney cultures from constitutive Scribble knockout animals. In summary these results indicate that basolateral polarity signaling via Scribble is dispensable for podocyte function, highlighting the unique feature of podocyte development with its significant apical membrane expansions being dominated by apical polarity complexes rather than by basolateral polarity signaling.     

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 β1, 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.     

Suppression of Rapidly Progressive Mouse Glomerulonephritis with the Non-Steroidal Mineralocorticoid Receptor Antagonist BR-4628

Background/Aim

Steroidal mineralocorticoid receptor antagonists (MRAs) are effective in the treatment of kidney disease; however, the side effect of hyperkalaemia, particularly in the context of renal impairment, is a major limitation to their clinical use. Recently developed non-steroidal MRAs have distinct characteristics suggesting that they may be superior to steroidal MRAs. Therefore, we explored the benefits of a non-steroidal MRA in a model of rapidly progressive glomerulonephritis.

Methods

Accelerated anti-glomerular basement membrane (GBM) glomerulonephritis was induced in groups of C57BL/6J mice which received no treatment, vehicle or a non-steroidal MRA (BR-4628, 5mg/kg/bid) from day 0 until being killed on day 15 of disease. Mice were examined for renal injury.

Results

Mice with anti-GBM glomerulonephritis which received no treatment or vehicle developed similar disease with severe albuminuria, impaired renal function, glomerular tuft damage and crescents in 40% of glomeruli. In comparison, mice which received BR-4628 displayed similar albuminuria, but had improved renal function, reduced severity of glomerular tuft lesions and a 50% reduction in crescents. The protection seen in BR-4628 treated mice was associated with a marked reduction in glomerular macrophages and T-cells and reduced kidney gene expression of proinflammatory (CCL2, TNF-α, IFN-γ) and profibrotic molecules (collagen I, fibronectin). In addition, treatment with BR-4626 did not cause hyperkalaemia or increase urine Na+/K+ excretion (a marker of tubular dysfunction).

Conclusions

The non-steroidal MRA (BR-4628) provided substantial suppression of mouse crescentic glomerulonephritis without causing tubular dysfunction. This finding warrants further investigation of non-steroidal MRAs as a therapy for inflammatory kidney diseases.     

Glomerular podocytes in cultured rat kidney slices

Summary The ultrastructure of rat glomerular epithelial cells (podocytes) in kidney slices in vitro was examined using qualitative and quantitative electron microscopy. The kidney slices were cultured in Medium 199 with Hanks' salts in a 5% CO₂/95% O₂ environment for up to 14 days. Few changes in podocyte ultrastructure occurred in the first 12 h of culture, but by 24 h cell bodies were rounded, microvilli were present on all podocyte surfaces, and some foot processes had been replaced by flattened expanses of cytoplasm. These changes were more pronounced by 3 days, when some podocytes had developed pseudopodal extensions and appeared to be migrating from glomeruli onto the slice surface. Podocytes could still be identified after 8, 10 and 14 days of culture, although relatively few glomeruli remained at 14 days. Morphometric methods were used to analyse podocyte shape, volume and surface area during the first 4 days of culture. The most significant change involved loss of foot processes: the number of filtration slits per 100 m of basement membrane decreased from 211.8 ± 15.0 (mean ± SD) at the commencement of culture, to 55.3 ± 22.6 after 2 days (P < 0.001). These data provide baseline information for in vitro studies on the effects of nephrotoxins on podocytes.     

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.     

Ligation of α-Dystroglycan on Podocytes Induces Intracellular Signaling: A New Mechanism for Podocyte Effacement?

Background

α-Dystroglycan is a negatively charged glycoprotein that covers the apical and basolateral membrane of the podocyte. Its transmembrane binding to the cytoskeleton is regulated via tyrosine phosphorylation (pY892) of β-dystroglycan. At the basolateral side α-dystroglycan binds the glomerular basement membrane. At the apical membrane, it plays a role in the maintenance of the filtration slit. In this study, we evaluated whether ligation of α-dystroglycan with specific antibodies or natural ligands induces intracellular signaling, and whether there is an effect on podocyte architecture.

Methodology/Principal Findings

Conditionally immortalized podocytes were exposed in vitro to antibodies to α-dystroglycan, and to fibronectin, biglycan, laminin and agrin. Intracellular calcium fluxes, phosphorylation of β-dystroglycan and podocyte architecture were studied. Antibodies to α-dystroglycan could specifically induce calcium signaling. Fibronectin also induced calcium signaling, and led to dephosphorylation of pY892 in β-dystroglycan. Ligation of α-dystroglycan resulted in an altered actin architecture, a decreased number of podocyte pedicles and a more flattened appearance of the podocyte.

Conclusions/Significance

We conclude that ligation of α-dystroglycan on podocytes induces intracellular calcium signaling, which leads to an altered cytoskeleton architecture akin to the situation of foot process effacement. In particular the ability of fibronectin to induce intracellular signaling events is of interest, since the expression and excretion of this protein is upregulated in several proteinuric diseases. Therefore, fibronectin-induced signaling via dystroglycan may be a novel mechanism for foot process effacement in proteinuric diseases.     

Ultrastructure of the coxal gland of the horseshoe crab Limulus polyphemus: Evidence for ultrafiltration and osmoregulation

Electron microscopic examination of the paired coxal glands of the horseshoe crab Limulus polyphemus, focusing on urinary and vascular channels, shows six morphologically distinct regions. Each of four nephridial lobes consists of two cortical layers surrounding a medulla. The outer and inner cortexes contain blood vessels separated by a basement membrane from the urinary space lined by podocytes. Podocyte foot processes are applied to the basement membrane, interdigitate with those from other podocytes, and have a filtration slit diaphragm between them. Cortical morphology demonstrates ultrafiltration of blood, a previously undescribed function of the gland, as well as possible endocytic reabsorption of materials by the podocytes. The medulla drains into the stolon connecting the four lobes. These two areas have urinary tubules of cuboidal epithelium featuring microvillous-like apical projections, cytoplasmic vesicles and vacuoles, elaborate lateral interdigitations with septate junctions, and basal invaginations containing numerous mitochondria. These tubules are closely surrounded by blood channels, lined by a basement membrane containing embedded support cells. The medulla and stolon morphology are suggestive of both ion transport and water movement, in keeping with the gland's role in osmoregulation. The stolon empties into the end sac in the base of the most posterior lobe. It is lined by tall epithelium exhibiting apical overlap, blunt projections into the lumen, apparent endocytic vesicles, and basal plasma membrane infoldings with mitochondria. The end sac drains into the conducting nephric duct, the proximal end of which is lined by a cuticle. J. Morphol. 234:233–252, 1997. © 1997 Wiley-Liss, Inc.     

Electron microscopic study of glomerular filtration barrier in the rat kidney embedded in polymerized glutaraldehyde-urea.

Embedding kidney in polymerized glutaraldehyde-urea favors the retention of glycoprotein matrix of the cell coat and the basement membrane of the glomeruli. The basement membrane appears as a single layer with uniform amorphous matrix. Thick glycoprotein coat covers the whole surface of prodocytes and their foot processes. In areas other than the slits and the portion of the foot processes which touch on the basement membrane, the coat is a continuous layer with an average thickness of 490 A. In the slits between the foot processes of podocytes there is an actual fusion of glycoprotein coats; the average width of the slit is 415 A. The glycoprotein 'plugs' in the slit may be a significant portion of the glomerular filtration barrier against macromolecules, together with the basement membrane and the slit diaphragms.     

Formation of basement membrane in extracapillary proliferates in rapidly progressive glomerulonephritis.

In the extracapillary proliferations (crescents) of the glomeruli in glomerulonephritis, basement membranes appear and in addition "secretory bodies" are formed in the cisternae of the rough endoplasmatic reticulum. The findings permit the conclusion that proliferated visceral epithelial cells are involved in the crescent formation to a greater extent than previously assumed.     

Experimental study on developing rat kidney after Naja haje envenomation

In the present experiment 164 pregnant white Wistar rats were used to study the effect of Naja haje (Egyptian cobra) venom on the developing kidney. The rats were divided into 3 groups; a control group, a group receiving one LD50 of N. haje venom and the third injected with 1/8 of LD50. The injection was performed at different stages of gestation. After birth, the neonates of group I and III and embryos of group II were examined histologically, histochemically and electron-microscopically. Both lethal and sublethal doses of N. haje venom produced haemorrhages and vascular congestion of the developing kidney. The lethal dose had degenerative effects on the podocytes and endothelium. Tubular damage appeared mainly as mitochondrial degeneration and bud-like extension, protrusions of cytoplasm and vacuolization. The succinic dehydrogenase enzyme showed decreased activity. The sublethal dose had an effect on the glomerular basement membrane in the form of splitting, increased mesangial cells and matrix, mitochondrial degeneration and fusion of podocyte processes. Tubulization of the parietal epithelium, vacuolization of the proximal tubules, mitochondrial degeneration and apical budding were evident.     

Possible sites of ultrafiltration in Tubifex tubifex Müller (Annelida,Oligochaeta)

Summary The endothelia of Tubifex tubifex Müller consist of myoendothelial cells, chloragocytes, or podocytes. The latter seem to occur only as windows on the ventral vessel which has an endothelium of myoendothelial cells elsewhere. The podocytes are large cells, with several processes on the inner side which ramify into several pedicels. These are aligned upon the outside of the basement membrane which lines the inside of the endothelium. The gaps between adjacent pedicels are about 40 nm wide. In capillaries fenestrated endothelia occur with irregular spacings measuring up to 0.4–1 m. A diaphragm in podocytes or capillary fenestrations do not seem to exist. The basement membrane is the only continuous layer lining the blood vessels and capillaries of Tubifex with a rather uniform diameter in the range of 50 nm. It is the only permeability barrier between blood and coelomic fluid.     

Nephrotic syndrome and subepithelial deposits in a mouse model of immune-mediated anti-podocyte glomerulonephritis

Subepithelial immune complex deposition in glomerular disease causes local inflammation and proteinuria by podocyte disruption. A rat model of membranous nephropathy, the passive Heymann nephritis, suggests that Abs against specific podocyte Ags cause subepithelial deposit formation and podocyte foot process disruption. In this study, we present a mouse model in which a polyclonal sheep anti-mouse podocyte Ab caused subepithelial immune complex formation. Mice developed a nephrotic syndrome with severe edema, proteinuria, hypoalbuminemia, and elevated cholesterol and triglycerides. Development of proteinuria was biphasic: an initial protein loss was followed by a second massive increase of protein loss beginning at approximately day 10. By histology, podocytes were swollen. Electron microscopy revealed 60-80% podocyte foot process effacement and subepithelial deposits, but no disruption of the glomerular basement membrane. Nephrin and synaptopodin staining was severely disrupted, and podocyte number was reduced in anti-podocyte serum-treated mice, indicating severe podocyte damage. Immunohistochemistry detected the injected anti-podocyte Ab exclusively along the glomerular filtration barrier. Immunoelectron microscopy localized the Ab to podocyte foot processes and the glomerular basement membrane. Similarly, immunohistochemistry localized mouse IgG to the subepithelial space. The third complement component (C3) was detected in a linear staining pattern along the glomerular basement membrane and in the mesangial hinge region. However, C3-deficient mice were not protected from podocyte damage, indicating a complement-independent mechanism. Twenty proteins were identified as possible Ags to the sheep anti-podocyte serum by mass spectrometry. Together, these data establish a reproducible model of immune-mediated podocyte injury in mice with subepithelial immune complex formation.     

An electron microscope study of kidney basement membrane changes in the mouse by lipoteichoic acid from Streptococcus pyogenes

Mice injected repeatedly, intraperitoneally or intravenously, for approximately 1 month with a total of 1.04 mg lipoteichoic acid from a nephritogenic strain of Streptococcus pyogenes lost weight. Analysis by electron microscopy revealed that they also exhibited extensive kidney changes in basement membrane morphology which resembled, in part, those observed in human poststreptococcal glomerulonephritis. For example, the glomerular basement membrane became electron dense and exhibited at least a twofold increase in width sporadically within the same preparation after exposure to lipoteichoic acid. Also, whereas appreciable loss or reduction in epithelial foot processes as a result of fusion was clearly evident, epithelial slits and slit membranes or diaphragms between normal foot processes were not selectively affected. In addition, another mostly thickened, highly coiled or serpentinelike basement membrane with amorphous nodules appeared in these preparations. This type membrane was not observed surrounding the capillary lumina and was the most pronounced abnormality apparent in almost all preparations from mice exposed to lipoteichoic acid. Likewise, the proximal tubular basement membrane became variable in width and increased in electron density in mice given lipoteichoic acid as compared with controls. In addition, this membrane was often punctuated with various morphological protrusions originating from only its thickened areas and which extended away from, and not into, the capillary space. This change was only associated with the basement membrane of the proximal tubular capillaries. All membrane changes persisted but gradually subsided, with normal kidney membrane morphology reappearing on the 4th day following the last injection of lipoteichoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptor activator of NF-kappaB and podocytes: towards a function of a novel receptor-ligand pair in the survival response of podocyte injury

Background

Glomerulosclerosis correlates with reduction in podocyte number that occurs through mechanisms which include apoptosis. Podocyte injury or podocyte loss in the renal glomerulus has been proposed as the crucial mechanism in the development of glomerulosclerosis. However, the mechanism by which podocytes respond to injury is poorly understood. TNF and TNF receptor superfamilies are important in the pathogenesis of podocyte injury and apoptosis. The ligand of receptor activator of NF-kappaB (RANKL) and receptor activator of NF-kappaB (RANK) are members of the TNF and receptor superfamilies. We investigated whether RANK - RANKL is a receptor - ligand complex for podocytes responding to injury.

Methodology/Principal Findings

In this study, RANKL and RANK were examined in human podocyte diseases and a rat model of puromycin aminonucleoside nephrosis (PAN). Compared with controls, RANK and RANKL were increased in both human podocyte diseases and the rat PAN model; double immunofluorescence staining revealed that RANK protein expression was mainly attributed to podocytes. Immunoelectron microscopy showed that RANK was localized predominantly at the top of the foot process membrane and the cytoplasm of rat podocyte. In addition, RANK was upregulated in mouse podocytes in vitro after injury induced by puromycin aminonucleoside (PA). Knockdown of RANK expression by small interference RNA (siRNA) exacerbated podocyte apoptosis induced by PA. However, RANKL inhibited significantly the apoptosis of podocytes induced by PA.

Conclusions/Significance

These findings suggest the increase in RANK–RANKL expression is a response to podocyte injury, and RANK–RANKL may be a novel receptor–ligand complex for the survival response during podocyte injury.     

Altered renal morphology in transgenic mice with cholecystokinin overexpression

Although cholecystokinin is a regulatory peptide with a predominant role in the brain and the gastrointestinal tract, there is an increasing evidence for its role in the kidney. The aim of this study was to reveal morphological changes in the structure of kidney of mice with cholecystokinin overexpression by means of light, transmission and scanning electron microscope, and atomic force microscopy. Using immunohistochemistry the expression of important basement membrane proteins collagen IV, laminin and fibronectin, as well the distribution of cholecystokinin-8 in the renal structures was evaluated. The altered morphology of kidneys of mice with cholecystokinin overexpression was seen by all microscopic techniques used. The renal corpuscles were relatively small with narrow capsular lumen. The basement membranes of renal tubules were thickened and the epithelial cells were damaged, which was more pronounced for distal tubules. Characteristic feature was the increased number of vesicles seen throughout the epithelial cells of proximal and especially in distal tubules reflecting to the enhanced cellular degeneration. The relative expression of laminin but not collagen IV in the glomerular basement membrane was higher than in the tubular basement membranes. The content of fibronectin, in opposite, was higher in tubular membranes. Cholecystokinin-8 was clearly expressed in the glomeruli, in Bowman’s capsule, in proximal and distal tubules, and in collecting ducts. Ultrastructural studies showed irregularly thickened glomerular basement membranes to which elongated cytopodia of differently shaped podocytes were attached. As foot processes were often fused the number of filtration pores was decreased. In conclusion, cholecystokinin plays important role in renal structural formation and in functioning as different aspects of urine production in mice with cholecystokinin overexpression are affected-the uneven glomerular basement membrane thickening, structural changes in podocytes and in filtration slits affect glomerular filtration, while damaged tubular epithelial cells and changed composition of thickened tubular basement membranes affect reabsorption.     

The Golgi apparatus in the endomembrane-rich gastric parietal cells exist as functional stable mini-stacks dispersed throughout the cytoplasm

Background information. Acid‐secreting gastric parietal cells are polarized epithelial cells that harbour highly abundant and specialized, H⁺, K⁺ ATPase‐containing, tubulovesicular membranes in the apical cytoplasm. The Golgi apparatus has been implicated in the biogenesis of the tubulovesicular membranes; however, an unanswered question is how a typical Golgi organization could regulate normal membrane transport within the membrane‐dense cytoplasm of parietal cells. Results. Here, we demonstrate that the Golgi apparatus of parietal cells is not the typical juxta‐nuclear ribbon of stacks, but rather individual Golgi units are scattered throughout the cytoplasm. The Golgi membrane structures labelled with markers of both cis‐ and trans‐Golgi membrane, indicating the presence of intact Golgi stacks. The parietal cell Golgi stacks were closely aligned with the microtubule network and were shown to participate in both anterograde and retrograde transport pathways. Dispersed Golgi stacks were also observed in parietal cells from H⁺, K⁺ ATPase‐deficient mice that lack tubulovesicular membranes. Conclusions. These results indicate that the unusual organization of individual Golgi stacks dispersed throughout the cytoplasm of these terminally differentiated cells is likely to be a developmentally regulated event.     

Loss of the tumor suppressor Vhlh leads to upregulation of Cxcr4 and rapidly progressive glomerulonephritis in mice

Rapidly progressive glomerulonephritis (RPGN) is a clinical syndrome characterized by loss of renal function within days to weeks and by glomerular crescents on biopsy. The pathogenesis of this disease is unclear, but circulating factors are believed to have a major role. Here, we show that deletion of the Von Hippel-Lindau gene (Vhlh) from intrinsic glomerular cells of mice is sufficient to initiate a necrotizing crescentic glomerulonephritis and the clinical features that accompany RPGN. Loss of Vhlh leads to stabilization of hypoxia-inducible factor alpha subunits (HIFs). Using gene expression profiling, we identified de novo expression of the HIF target gene Cxcr4 (ref. 3) in glomeruli from both mice and humans with RPGN. The course of RPGN is markedly improved in mice treated with a blocking antibody to Cxcr4, whereas overexpression of Cxcr4 alone in podocytes of transgenic mice is sufficient to cause glomerular disease. Collectively, these results indicate an alternative mechanism for the pathogenesis of RPGN and glomerular disease in an animal model and suggest novel molecular pathways for intervention in this disease.     

Structure of the cumulus oophorus at the time of fertilization

Summary The paired external glomus of the fully developed pronephros has been studied in early larvae (ammocoetes) of 2 lamprey species, Lampetra fluviatilis and Petromyzon marinus, several weeks after hatching and newly hatched, by use of light-, scanning (SEM) and transmission (TEM) electron microscopy. Three weeks after hatching the glomus is a complex of capillary loops supplied by a single arteriole branching from the aorta. The glomus consists of 3 cell types: podocytes, fenestrated endothelium, and mesangial cells. A basement membrane, which has a close contact to the podocytes, is the only continuous barrier between blood and the coelomic cavity. The glomus exhibits all fine-structural elements known to be essential for function in the glomeruli of other vertebrates. We therefore assume the pronephric glomus of lampreys to be functional in ultrafiltration, with the ultrafiltrate released into the coelomic cavity. In newly hatched larvae, the structure of the glomus is not fully developed. In this earlier stage several afferent arterioles supply each glomus. The endothelial cells in the glomar capillaries still lack regular epithelial organization and resemble mesenchymal cells. However, the presence of typical podocytes stretching over a continuous basement membrane suggests that the tissue is already capable of ultrafiltration.This paper is dedicated to the memory of Professor W. Bargmann, long-time editor of Cell and Tissue Research, the author of a splendid review on the structure of the vertebrate kidney and a master of German scientific writing