Hyperglycemia - A culprit of podocyte pathology in the context of glycogen metabolism.

Prolonged disruption in the balance of glucose can result in metabolic disorders. The kidneys play a significant role in regulating blood glucose levels. However, when exposed to chronic hyperglycemia, the kidneys' ability to handle glucose metabolism may be impaired, leading to an accumulation of glycogen. Earlier studies have shown that there can be a significant increase in glucose storage in the form of glycogen in the kidneys in diabetes. Podocytes play a crucial role in maintaining the integrity of filtration barrier. In diabetes, exposure to elevated glucose levels can lead to significant metabolic and structural changes in podocytes, contributing to kidney damage and the development of diabetic kidney disease. The accumulation of glycogen in podocytes is not a well-established phenomenon. However, a recent study has demonstrated the presence of glycogen granules in podocytes. This review delves into the intricate connections between hyperglycemia and glycogen metabolism within the context of the kidney, with special emphasis on podocytes. The aberrant storage of glycogen has the potential to detrimentally impact podocyte functionality and perturb their structural integrity. This review provides a comprehensive analysis of the alterations in cellular signaling pathways that may potentially lead to glycogen overproduction in podocytes.

Pit 1 transporter (SLC20A1) as a key factor in the NPP1-mediated inhibition of insulin signaling in human podocytes.

Podocytes are crucially involved in blood filtration in the glomerulus. Their proper function relies on efficient insulin responsiveness. The insulin resistance of podocytes, defined as a reduction of cell sensitivity to this hormone, is the earliest pathomechanism of microalbuminuria that is observed in metabolic syndrome and diabetic nephropathy. In many tissues, this alteration is mediated by the phosphate homeostasis-controlling enzyme nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1). By binding to the insulin receptor (IR), NPP1 inhibits downstream cellular signaling. Our previous research found that hyperglycemic conditions affect another protein that is involved in phosphate balance, type III sodium-dependent phosphate transporter 1 (Pit 1). In the present study, we evaluated the insulin resistance of podocytes after 24 h of incubation under hyperinsulinemic conditions. Thereafter, insulin signaling was inhibited. The formation of NPP1/IR complexes was observed at that time. A novel finding in the present study was our observation of an interaction between NPP1 and Pit 1 after the 24 h stimulation of podocytes with insulin. After downregulation of the SLC20A1 gene, which encodes Pit 1, we established insulin resistance in podocytes that were cultured under native conditions, manifested as a lack of intracellular insulin signaling and the inhibition of glucose uptake via the glucose transporter type 4. These findings suggest that Pit 1 might be a major factor that participates in the NPP1-mediated inhibition of insulin signaling.

Role of lysosomes in insulin signaling and glucose uptake in cultured rat podocytes.

Podocytes are sensitive to insulin, which governs the functional and structural integrity of podocytes that are essential for proper function of the glomerular filtration barrier. Lysosomes are acidic organelles that are implicated in regulation of the insulin signaling pathway. Cathepsin D (CTPD) and lysosome-associated membrane protein 1 (LAMP1) are major lysosomal proteins that reflect the functional state of lysosomes. However, the effect of insulin on lysosome activity and role of lysosomes in the regulation of insulin-dependent glucose uptake in podocytes are unknown. Our studies showed that the short-term incubation of podocytes with insulin decreased LAMP1 and CTPD mRNA levels. Insulin and bafilomycin A1 reduced both the amounts of LAMP1 and CTPD proteins and activity of CTPD, which were associated with a decrease in the fluorescence intensity of lysosomes that were labeled with LysoTracker. Bafilomycin A1 inhibited insulin-dependent endocytosis of the insulin receptor and increased the amounts of the insulin receptor and glucose transporter 4 on the cell surface of podocytes. Bafilomycin A1 also inhibited insulin-dependent glucose uptake despite an increase in the amount of glucose transporter 4 in the plasma membrane of podocytes. These results suggest that lysosomes are signaling hubs that may be involved in the coupling of insulin signaling with the regulation of glucose uptake in podocytes. The dysregulation of this mechanism can lead to the dysfunction of podocytes and development of insulin resistance.

The Role of PKGIα and AMPK Signaling Interplay in the Regulation of Albumin Permeability in Cultured Rat Podocytes.

The permeability of the glomerular filtration barrier (GFB) is mainly regulated by podocytes and their foot processes. Protein kinase G type Iα (PKGIα) and adenosine monophosphate-dependent kinase (AMPK) affect the contractile apparatus of podocytes and influence the permeability of the GFB. Therefore, we studied the interplay between PKGIα and AMPK in cultured rat podocytes. The glomerular permeability to albumin and transmembrane FITC-albumin flux decreased in the presence of AMPK activators and increased in the presence of PKG activators. The knockdown of PKGIα or AMPK with small-interfering RNA (siRNA) revealed a mutual interaction between PKGIα and AMPK and influenced podocyte permeability to albumin. Moreover, PKGIα siRNA activated the AMPK-dependent signaling pathway. AMPKα2 siRNA increased basal levels of phosphorylated myosin phosphate target subunit 1 and decreased the phosphorylation of myosin light chain 2. Podocytes that were treated with AMPK or PKG activators were characterized by the different organization of actin filaments within the cell. Our findings suggest that mutual interactions between PKGIα and AMPKα2 regulate the contractile apparatus and permeability of the podocyte monolayer to albumin. Understanding this newly identified molecular mechanism in podocytes provides further insights into the pathogenesis of glomerular disease and novel therapeutic targets for glomerulopathies.

Hyperglycemic environment disrupts phosphate transporter function and promotes calcification processes in podocytes and isolated glomeruli.

Soft tissue calcification is a pathological phenomenon that often occurs in end-stage chronic kidney disease (CKD), which is caused by diabetic nephropathy, among other factors. Hyperphosphatemia present during course of CKD contributes to impairments in kidney function, particularly damages in the glomerular filtration barrier (GFB). Essential elements of the GFB include glomerular epithelial cells, called podocytes. In the present study, we found that human immortalized podocytes express messenger RNA and protein of phosphate transporters, including NaPi 2c (SLC34A3), Pit 1 (SLC20A1), and Pit 2 (SLC20A2), which are sodium-dependent and mediate intracellular phosphate (Pi) transport, and XPR1, which is responsible for extracellular Pi transport. We found that cells that were grown in a medium with a high glucose (HG) concentration (30 mM) expressed less Pit 1 and Pit 2 protein than podocytes that were cultured in a standard glucose medium (11 mM). We found that exposure of the analyzed transporters in the cell membrane of the podocyte is altered by HG conditions. We also found that the activity of tissue nonspecific alkaline phosphatase increased in HG, causing a rise in Pi generation. Additionally, HG led to a reduction of the amount of ectonucleotide pyrophosphatase/phosphodiesterase 1 in the cell membrane of podocytes. The extracellular concentration of pyrophosphate also decreased under HG conditions. These data suggest that a hyperglycemic environment enhances the production of Pi in podocytes and its retention in the extracellular space, which may induce glomerular calcification.

Hyperglycemia alters mitochondrial respiration efficiency and mitophagy in human podocytes.

Podocytes constitute the outer layer of the renal glomerular filtration barrier. Their energy requirements strongly depend on efficient oxidative respiration, which is tightly connected with mitochondrial dynamics. We hypothesized that hyperglycemia modulates energy metabolism in glomeruli and podocytes and contributes to the development of diabetic kidney disease. We found that oxygen consumption rates were severely reduced in glomeruli from diabetic rats and in human podocytes that were cultured in high glucose concentration (30 mM; HG). In these models, all of the mitochondrial respiratory parameters, including basal and maximal respiration, ATP production, and spare respiratory capacity, were significantly decreased. Podocytes that were treated with HG showed a fragmented mitochondrial network, together with a decrease in expression of the mitochondrial fusion markers MFN1, MFN2, and OPA1, and an increase in the activity of the fission marker DRP1. We showed that markers of mitochondrial biogenesis, such as PGC-1α and TFAM, decreased in HG-treated podocytes. Moreover, PINK1/parkin-dependent mitophagy was inhibited in these cells. These results provide evidence that hyperglycemia impairs mitochondrial dynamics and turnover, which may underlie the remarkable deterioration of mitochondrial respiration parameters in glomeruli and podocytes.

The PKGIα-Rac1 pathway is a novel regulator of insulin-dependent glucose uptake in cultured rat podocytes.

Insulin plays a major role in regulating glucose homeostasis in podocytes. Protein kinase G type Iα (PKGIα) plays an important role in regulating glucose uptake in these cells. Rac1 signaling plays an essential role in the reorganization of the actin cytoskeleton and is also essential for insulin-stimulated glucose transport. The experiments were conducted using primary rat podocytes. We performed western blot analysis, evaluated small GTPases activity assays, measured radioactive glucose uptake, and performed immunofluorescence imaging to analyze the role of PKGIα-Rac1 signaling in regulating podocyte function. We also utilized a small-interfering RNA-mediated approach to determine the role of PKGIα and Rac1 in regulating glucose uptake in podocytes. The present study investigated the influence of the PKGI pathway on the insulin-dependent regulation of activity and cellular localization of small guanosine triphosphatases in podocytes. We found that the PKGIα-dependent activation of Rac1 signaling induced activation of the PAK/cofilin pathway and increased insulin-mediated glucose uptake in podocytes. The downregulation of PKGIα or Rac1 expression abolished this effect. Rac1 silencing prevented actin remodeling and GLUT4 translocation close to the cell membrane. These data provide evidence that PKGIα-dependent activation of the Rac1 signaling pathways is a novel regulator of insulin-mediated glucose uptake in cultured rat podocytes.

Insulin resistance in glomerular podocytes: Potential mechanisms of induction.

Glomerular podocytes are a target for the actions of insulin. Accumulating evidence indicates that exposure to nutrient overload induces insulin resistance in these cells, manifested by abolition of the stimulatory effect of insulin on glucose uptake. Numerous recent studies have investigated potential mechanisms of the induction of insulin resistance in podocytes. High glucose concentrations stimulated reactive oxygen species production through NADPH oxidase activation, decreased adenosine monophosphate-activated protein kinase (AMPK) phosphorylation, and reduced deacetylase sirtuin 1 (SIRT1) protein levels and activity. Calcium signaling involving transient receptor potential cation channel C, member 6 (TRPC6) also was demonstrated to play an essential role in the regulation of insulin-dependent signaling and glucose uptake in podocytes. Furthermore, podocytes exposed to diabetic environment, with elevated insulin levels become insulin resistant as a result of degradation of insulin receptor (IR), resulting in attenuation of insulin signaling responsiveness. Also elevated levels of palmitic acid appear to be an important factor and contributor to podocytes insulin resistance. This review summarizes cellular and molecular alterations that contribute to the development of insulin resistance in glomerular podocytes.

Purinergic P2 receptors: Involvement and therapeutic implications in diabetes-related glomerular injury.

The purinergic activation of P2 receptors initiates a powerful and rapid signaling cascade that contributes to the regulation of an array of physiological and pathophysiological processes in many organs, including the kidney. P2 receptors are broadly distributed in both epithelial and vascular renal cells. Disturbances of purinergic signaling can lead to impairments in renal function. A growing body of evidence indicates changes in P2 receptor expression and nucleotide metabolism in chronic renal injury and inflammatory diseases. Increasing attention has focused on purinergic P2X7 receptors, which are not normally expressed in healthy kidney tissue but are highly expressed at sites of tissue damage and inflammation. Under hyperglycemic conditions, several mechanisms that are linked to purinergic signaling and involve nucleotide release and degradation are disrupted, resulting in the accumulation of adenosine 5'-triphosphate in the bloodstream in diabetes. Dysfunction of the purinergic system might be associated with serious vascular complications in diabetes, including diabetic nephropathy. This review summarizes our current knowledge of the role of P2 receptors in diabetes-related glomerular injury and its implications for new therapeutics for diabetic nephropathy.

Involvement of nitric oxide synthase/nitric oxide pathway in the regulation of SIRT1-AMPK crosstalk in podocytes: Impact on glucose uptake.

The protein deacetylase sirtuin 1 (SIRT1) and adenosine monophosphate-dependent protein kinase (AMPK) play important roles in the development of insulin resistance. In glomerular podocytes, crosstalk between these two enzymes may be altered under hyperglycemic conditions. SIRT1 protein levels and activity and AMPK phosphorylation decrease under hyperglycemic conditions, with concomitant inhibition of the effect of insulin on glucose uptake into these cells. Nitric oxide (NO)-dependent regulatory signaling pathways have been shown to be downregulated under diabetic conditions. The present study examined the involvement of the NO synthase (NOS)/NO pathway in the regulation of SIRT1-AMPK signaling and glucose uptake in podocytes. We examined the effects of NOS/NO pathway alterations on SIRT1/AMPK signaling and glucose uptake using pharmacological tools and a small-interfering transfection approach. We also examined the ability of the NOS/NO pathway to protect podocytes against high glucose-induced alterations of SIRT1/AMPK signaling and insulin-dependent glucose uptake. Inhibition of the NOS/NO pathway reduced SIRT1 protein levels and activity, leading to a decrease in AMPK phosphorylation and blockade of the effect of insulin on glucose uptake. Treatment with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) prevented high glucose-induced decreases in SIRT1 and AMPK activity and increased GLUT4 protein expression, thereby improving glucose uptake in podocytes. These findings suggest that inhibition of the NOS/NO pathway may result in alterations of the effects of insulin on glucose uptake in podocytes. In turn, the enhancement of NOS/NO pathway activity may prevent these deleterious effects of high glucose concentrations, thus bidirectionally stimulating the SIRT1-AMPK reciprocal activation loop.

Role of Klotho in Hyperglycemia: Its Levels and Effects on Fibroblast Growth Factor Receptors, Glycolysis, and Glomerular Filtration.

Hyperglycemic conditions (HG), at early stages of diabetic nephropathy (DN), cause a decrease in podocyte numbers and an aberration of their function as key cells for glomerular plasma filtration. Klotho protein was shown to overcome some negative effects of hyperglycemia. Klotho is also a coreceptor for fibroblast growth factor receptors (FGFRs), the signaling of which, together with a proper rate of glycolysis in podocytes, is needed for a proper function of the glomerular filtration barrier. Therefore, we measured levels of Klotho in renal tissue, serum, and urine shortly after DN induction. We investigated whether it influences levels of FGFRs, rates of glycolysis in podocytes, and albumin permeability. During hyperglycemia, the level of membrane-bound Klotho in renal tissue decreased, with an increase in the shedding of soluble Klotho, its higher presence in serum, and lower urinary excretion. The addition of Klotho increased FGFR levels, especially FGFR1/FGFR2, after their HG-induced decrease. Klotho also increased levels of glycolytic parameters of podocytes, and decreased podocytic and glomerular albumin permeability in HG. Thus, we found that the decrease in the urinary excretion of Klotho might be an early biomarker of DN and that Klotho administration may have several beneficial effects on renal function in DN.

Regulation of podocytes function by AMP-activated protein kinase.

Podocytes are unique, highly specialized, terminally differentiated cells that form an essential, integral part of the glomerular filter. These cells limit the outside border of the glomerular basement membrane, forming a tight barrier that prevents significant protein loss from the capillary space. The slit diaphragm formed by podocytes is crucial for maintaining glomerular integrity and function. They are the target of injury in many glomerular diseases, including hypertension and diabetes mellitus. Accumulating studies have revealed that AMP-activated protein kinase (AMPK), an essential cellular energy sensor, might play a fundamental role in regulating podocyte metabolism and function. AMPK participates in insulin signaling, therefore controls glucose uptake and podocytes insulin sensitivity. It is also involved in insulin-dependent cytoskeleton reorganization in podocytes, mediating glomerular albumin permeability. AMPK plays an important role in the regulation of autophagy/apoptosis processes, which influence podocytes viability. The present review aimed to highlight the molecular mechanisms associated with AMPK that are involved in the regulation of podocyte function in health and disease states.

Extracellular ATP modulates podocyte function through P2Y purinergic receptors and pleiotropic effects on AMPK and cAMP/PKA signaling pathways.

Podocytes and their foot processes interlinked by slit diaphragms, constitute a continuous outermost layer of the glomerular capillary and seem to be crucial for maintaining the integrity of the glomerular filtration barrier. Purinergic signaling is involved in a wide range of physiological processes in the renal system, including regulating glomerular filtration. We evaluated the role of nucleotide receptors in cultured rat podocytes using non-selective P2 receptor agonists and agonists specific for the P2Y1, P2Y2, and P2Y4 receptors. The results showed that extracellular ATP evokes cAMP-dependent pathways through P2 receptors and influences remodeling of the podocyte cytoskeleton and podocyte permeability to albumin via coupling with RhoA signaling. Our findings highlight the relevance of the P2Y4 receptor in protein kinase A-mediated signal transduction to the actin cytoskeleton. We observed increased cAMP concentration and decreased RhoA activity after treatment with a P2Y4 agonist. Moreover, protein kinase A inhibitors reversed P2Y4-induced changes in RhoA activity and intracellular F-actin staining. P2Y4 stimulation resulted in enhanced AMPK phosphorylation and reduced reactive oxygen species generation. Our findings identify P2Y-PKA-RhoA signaling as the regulatory mechanism of the podocyte contractile apparatus and glomerular filtration. We describe a protection mechanism for the glomerular barrier linked to reduced oxidative stress and reestablished energy balance.

The TRPC6-AMPK Pathway is Involved in Insulin-Dependent Cytoskeleton Reorganization and Glucose Uptake in Cultured Rat Podocytes.

BACKGROUND/AIMS: Podocytes are dynamic polarized cells on the surface of glomerular capillaries that are an essential part of the glomerular filtration barrier. AMP-activated protein kinase (AMPK), a key regulator of glucose and fatty acid metabolism, plays a major role in obesity and type 2 diabetes. Accumulating evidence suggests that TRPC6 channels are crucial mediators of calcium transport in podocytes and are involved in regulating glomerular filtration. Here we investigated whether the AMPK-TRPC6 pathway is involved in insulin-dependent cytoskeleton reorganization and glucose uptake in cultured rat podocytes. METHODS: Western blot and immunofluorescence analysis confirmed AMPKα and TRPC6 expression, the phosphorylation of proteins associated with actin cytoskeleton reorganization (PAK, rac1, and cofilin), and the expression of insulin signaling proteins (Akt, Insulin receptor). Coimmunoprecipitation and immunofluorescence results demonstrated AMPKα/TRPC6 interaction. To ask whether TRPC6 is involved in the insulin regulation of glucose transport, we measured insulin-dependent (1, 2-3H)-deoxy-D-glucose uptake into podocytes after reducing TRPC6 activity pharmacologically and biochemically (TRPC6 siRNA). RESULTS: The results suggested a key role for the TRPC6 channel in the mediation of insulin-dependent activation of AMPKα2 and glucose uptake. Moreover, AMPK and TRPC6 activation were required to stimulate the Rac1 signaling pathway. CONCLUSION: These results suggest a potentially important new mechanism that regulates glucose transport in podocytes and that could be injurious during diabetes.

Metformin overcomes high glucose-induced insulin resistance of podocytes by pleiotropic effects on SIRT1 and AMPK.

Podocyte insulin sensitivity is critical for glomerular function, and the loss of appropriate insulin signaling leads to alterations and disorders featuring diabetic nephropathy. Energy-sensing pathways, such as AMP-dependent protein kinase (AMPK) and protein deacetylase SIRT1, have been shown to play an important role in insulin resistance. The absence of a stimulating effect of insulin on glucose uptake into podocytes after exposure to hyperglycemic conditions has been demonstrated to be related to a decreased level and activity of SIRT1 protein, leading to reduced AMPK phosphorylation. The present work was undertaken to investigate metformin's ability to restore the insulin responsiveness of podocytes by regulating SIRT1 and AMPK activities. Primary rat podocytes cultured with standard or high glucose concentrations for 5days were transfected with siRNAs targeting SIRT1, AMPKα1, or AMPKα2. SIRT1 activity was measured by a fluorometric method. Insulin-stimulated changes in glucose uptake were used to detect insulin resistance. Podocyte permeability was measured by a transmembrane albumin flux assay to examine podocytes functioning. Our results demonstrated that metformin activated SIRT1 and AMPK, prevented hyperglycemia-induced reduction of SIRT1 protein levels, ameliorated glucose uptake into podocytes, and decreased glomerular filtration barrier permeability. Furthermore, metformin activated AMPK in a SIRT1-independent manner, as the increase in AMPK phosphorylation after metformin treatment was not affected by SIRT1 downregulation. Therefore, the potentiating effect of metformin on insulin-resistant podocytes seemed to be dependent on AMPK, as well as SIRT1 activity, establishing multilateral effects of metformin action.

Insulin increases filtration barrier permeability via TRPC6-dependent activation of PKGIα signaling pathways.

Podocytes are dynamic polarized cells on the surface of glomerular capillaries and an essential component of the glomerular filtration barrier. Insulin increases the activation of protein kinase G type Iα (PKGIα) subunits, leading to podocyte dysfunction. In addition, accumulating evidence suggests that TRPC6 channels are crucial mediators of podocyte calcium handling and involved in the regulation of glomerular filtration. Therefore, we investigated whether TRPC6 is involved in the regulation of filtration barrier permeability by insulin via the PKGIα-dependent manner. TRPC channel inhibitor SKF96365 abolished insulin-dependent glomerular albumin permeability and transepithelial albumin flux in cultured rat podocytes. Insulin-evoked albumin permeability across podocyte monolayers was also blocked using TRPC6 siRNA. The effect of insulin on albumin permeability was mimicked by treating podocytes with TRPC channel activator (oleolyl-2-acetyl-sn-glycerol, OAG). Insulin or OAG treatment rapidly increased the superoxide generation through activation of NADH oxidase. TRPC inhibitor SKF96365 or siRNA knockdown of TRPC6 attenuated insulin-dependent increase of ROS production. Furthermore, TRPC inhibitor or downregulation of TRPC6 blocked insulin-induced rearrangement of the actin cytoskeleton and attenuated oxidative activation of PKGIα and changes in the phosphorylation of PKG target proteins MYPT1 and MLC. Moreover insulin regulated the PKGIα interaction with TRPC6 in cultured rat podocytes. Taken together, our data suggest a key role of TRPC6 channels in the mediation of insulin-dependent activation of PKGIα signaling pathways. Overall, we have identified a potentially important mechanism that may explain disturbances in filtration barrier permeability in many diseases with increased expression of TRPC6 and chronic Ca2+ overload.

Viability of primary cultured podocytes is associated with extracellular high glucose-dependent autophagy downregulation.

Structural and functional impairment of podocytes plays an important role in the development of diabetic nephropathy, a chronic complication of diabetes mellitus and leading cause of renal failure requiring renal replacement therapy. Autophagy plays a crucial role in podocyte viability and function, and its activity is modulated by a variety of pathophysiological factors found in diabetic milieu. Here we show that downregulation of autophagy is critical for podocyte survival in hyperglycemic environment. Moreover, long-term exposure to high glucose leads to inhibition of autophagy as well as to the development of insulin resistance in podocytes. Furthermore, impairment of autophagy is involved in alteration of insulin-dependent glucose uptake in podocytes, suggesting a relationship between these two processes. Taken together, our findings suggest that downregulation of podocyte autophagy, observed after long-term exposure to high glucose, results from their suppressed sensitivity to insulin, and may therefore lead to diminished podocyte cell viability as well as their reduced number in glomerulus.

SIRT1-AMPK crosstalk is involved in high glucose-dependent impairment of insulin responsiveness in primary rat podocytes.

Growing evidence indicates that in diabetes, high glucose concentrations affect podocyte metabolism and function. The crucial pathological feature of type 2 diabetes mellitus and metabolic syndrome is insulin resistance, often developed as a result of dysregulation of nutrient-responsible systems and disturbance of cellular homeostasis under diabetic conditions. Here, we report the involvement of the reciprocal interplay between deacetylase SIRT1 and protein kinase AMPK in podocyte high glucose-induced abolition of insulin-dependent glucose uptake, manifesting insulin resistance. Experiments were performed on primary rat podocytes cultured in standard or high glucose conditions. Immunodetection methods were used to determine SIRT1 protein level and AMPK phosphorylation degree. Insulin-stimulated changes in glucose uptake were used to determine podocyte responsiveness to insulin. SIRT1 activity was modulated by resveratrol, EX-527, or small interfering RNA targeting SIRT1. We have demonstrated that the absence of the stimulating effect of insulin on glucose uptake into primary rat podocytes after long-time exposition to high glucose concentrations, is a result of decreased SIRT1 protein levels and activity, associated with decreased AMPK phosphorylation degree, presumably underlying the induction of insulin resistance. Our findings suggest that the interplay between SIRT1 and AMPK is involved in the regulation of insulin action in podocytes.

Insulin increases glomerular filtration barrier permeability through PKGIα-dependent mobilization of BKCa channels in cultured rat podocytes.

Podocytes are highly specialized cells that wrap around glomerular capillaries and comprise a key component of the glomerular filtration barrier. They are uniquely sensitive to insulin; like skeletal muscle and fat cells, they exhibit insulin-stimulated glucose uptake and express glucose transporters. Podocyte insulin signaling is mediated by protein kinase G type I (PKGI), and it leads to changes in glomerular permeability to albumin. Here, we investigated whether large-conductance Ca²âº-activated K⁺ channels (BKCa) were involved in insulin-mediated, PKGIα-dependent filtration barrier permeability. Insulin-induced glomerular permeability was measured in glomeruli isolated from Wistar rats. Transepithelial albumin flux was measured in cultured rat podocyte monolayers. Expression of BKCa subunits was detected by RT-PCR. BKCa, PKGIα, and upstream protein expression were examined in podocytes with Western blotting and immunofluorescence. The BKCa-PKGIα interaction was assessed with co-immunoprecipitation. RT-PCR showed that primary cultured rat podocytes expressed mRNAs that encoded the pore-forming α subunit and four accessory ß subunits of BKCa. The BKCa inhibitor, iberiotoxin (ibTX), abolished insulin-dependent glomerular albumin permeability and PKGI-dependent transepithelial albumin flux. Insulin-evoked albumin permeability across podocyte monolayers was also blocked with BKCa siRNA. Moreover, ibTX blocked insulin-induced disruption of the actin cytoskeleton and changes in the phosphorylation of PKG target proteins, MYPT1 and RhoA. These results indicated that insulin increased filtration barrier permeability through mobilization of BKCa channels via PKGI in cultured rat podocytes. This molecular mechanism may explain podocyte injury and proteinuria in diabetes.

Extracellular purines' action on glomerular albumin permeability in isolated rat glomeruli: insights into the pathogenesis of albuminuria.

Purinoceptors (adrengeric receptors and P2 receptors) are expressed on the cellular components of the glomerular filtration barrier, and their activation may affect glomerular permeability to albumin, which may ultimately lead to albuminuria, a well-established risk factor for the progression of chronic kidney disease and development of cardiovascular diseases. We investigated the mechanisms underlying the in vitro and in vivo purinergic actions on glomerular filter permeability to albumin by measuring convectional albumin permeability (Palb) in a single isolated rat glomerulus based on the video microscopy method. Primary cultured rat podocytes were used for the analysis of Palb, cGMP accumulation, PKG-Iα dimerization, and immunofluorescence. In vitro, natural nucleotides (ATP, ADP, UTP, and UDP) and nonmetabolized ATP analogs (2-meSATP and ATP-γ-S) increased Palb in a time- and concentration-dependent manner. The effects were dependent on P2 receptor activation, nitric oxide synthase, and cytoplasmic guanylate cyclase. ATP analogs significantly increased Palb, cGMP accumulation, and subcortical actin reorganization in a PKG-dependent but nondimer-mediated route in cultured podocytes. In vivo, 2-meSATP and ATP-γ-S increased Palb but did not significantly affect urinary albumin excretion. Both agonists enhanced the clathrin-mediated endocytosis of albumin in podocytes. A product of adenine nucleotides hydrolysis, adenosine, increased the permeability of the glomerular barrier via adrenergic receptors in a dependent and independent manner. Our results suggest that the extracellular nucleotides that stimulate an increase of glomerular Palb involve nitric oxide synthase and cytoplasmic guanylate cyclase with actin reorganization in podocytes.