Agnieszka Piwkowska

Metformin induces suppression of NAD(P)H oxidase activity in podocytes

Hyperglycemia increases the production of reactive oxygen species (ROS). NAD(P)H oxidase, producing superoxide anion, is the main source of ROS in diabetic podocytes and their production contributes to the development of diabetic nephropathy. We have investigated the effect of an antidiabetic drug, metformin on the production of superoxide anion in cultured podocytes and attempted to elucidate underlying mechanisms. The experiments were performed in normal (NG, 5.6mM) and high (HG, 30mM) glucose concentration. Overall ROS production was measured by fluorescence of a DCF probe. Activity of NAD(P)H oxidase was measured by chemiluminescence method. The AMP-dependent kinase (AMPK) activity was determined by immunobloting, measuring the ratio of phosphorylated AMPK to total AMPK. Glucose accumulation was measured using 2-deoxy-[1,2-(3)H]-glucose. ROS production increased by about 27% (187+/-8 vs. 238+/-9 arbitrary units AU, P<0.01) in HG. Metformin (2mM, 2h) markedly reduced ROS production by 45% in NG and 60% in HG. Metformin decreased NAD(P)H oxidase activity in NG (36%) and HG (86%). AMPK activity was increased by metformin in NG and HG (from 0.58+/-0.07 to. 0.99+/-0.06, and from 0.53+/-0.03 to 0.64+/-0.03; P<0.05). The effects of metformin on the activities of NAD(P)H oxidase and AMPK were abolished in the presence of AMPK inhibitor, compound C. We have shown that metformin decreases production of ROS through reduction of NAD(P)H oxidase activity. We also have demonstrated relationship between activity of NAD(P)H oxidase and AMPK.

High glucose concentration affects the oxidant‐antioxidant balance in cultured mouse podocytes

Hyperglycemia is well‐recognized and has long‐term complications in diabetes mellitus and diabetic nephropathy. In podocytes, the main component of the glomerular barrier, overproduction of reactive oxygen species (ROS) in the presence of high glucose induces dysfunction and increases excretion of albumin in urine. This suggests an impaired antioxidant defense system has a role in the pathogenesis of diabetic nephropathy. We studied expression of NAD(P)H oxidase subunits by Western blotting and immunofluorescence and the activities of the oxidant enzyme, NAD(P)H, and antioxidant enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT), in mouse podocytes cultured in a high glucose concentration (30 mM). We found long‐term (3 and 5 days) exposure of mouse podocytes to high glucose concentrations caused oxidative stress, as evidenced by increased expression of Nox4 and activities of NAD(P)H oxidase (Δ 182%) and SOD (Δ 39%) and decreased activities of GPx (Δ −40%) and CAT (Δ −35%). These biochemical changes were accompanied by a rise in intracellular ROS production and accumulation of hydrogen peroxide in extracellular space. The role of Nox4 in ROS generation was confirmed with Nox4 siRNA. In conclusion, high glucose concentration affects the oxidant–antioxidant balance in mouse podocytes, resulting in enhanced generation of superoxide anions and its attenuated metabolism. These observations suggest free radicals may play an important role in the pathogenesis of diabetic nephropathy. J. Cell. Biochem. 112: 1661–1672, 2011.

Hydrogen peroxide induces dimerization of protein kinase G type Iα subunits and increases albumin permeability in cultured rat podocytes

Podocytes help regulate filtration barrier permeability in the kidneys. They express contractile proteins that are characteristic of smooth muscle cells as well as receptors for vasoactive factors such as angiotensin II and atrial natriuretic peptide (ANP). The later one generates intracellular cGMP, with subsequent activation of cGMP‐dependent protein kinase; PKG (isoform PKGIα and PKGIβ). In this study, we asked whether hydrogen peroxide (H2O2), a physiological vasorelaxing factor, affected podocyte permeability and the podoctye PKGIα signaling pathway. Expression of PKGIα was confirmed in cultured rat podocytes using RT‐PCR, immunofluorescence, and Western blotting. Exposure of podocytes to exogenous H2O2 (100 µM) in non‐reducing conditions increased the formation of PKGIα interprotein disulfide bonds, affected the phosphorylation of PKG target proteins, namely MYPT1 (maximal increase of about 57% at 30 min) and MLC (maximal decrease of about 62% at 10 min). Furthermore, H2O2 increased the permeability of a layer of podocytes to albumin: Transmembrane flux for albumin increased five‐fold (106.6 ± 5.2 µg/ml vs. 20.2 ± 2.5 µg/ml, P < 0.05, n = 5), and the PKG inhibitor Rp‐8‐Br‐cGMPS (100 µM) prevented the flux increase. These data suggest that oxidative modulation of PKGIα in podocytes plays an important role in the regulation of filtration barrier permeability. J. Cell. Physiol. 227: 1004–1016, 2012.

Insulin increases glomerular filtration barrier permeability through dimerization of protein kinase G type Iα subunits

The increase in the permeability of the glomerular barrier filtration to albumin is a well-known feature of diabetic microvasculature and a negative prognostic factor for vascular complications. However, the underlying mechanisms are incompletely understood. We demonstrated recently that superoxide anion generation increases dimerization of protein kinase G type Iα (PKGIα) subunits, leading to podocyte dysfunction. Here we investigated whether high insulin concentration is involved in PKGI-dependent hyperpermeability of the diabetic glomerular filtration barrier. We assessed changes in insulin-induced glomerular permeability by measuring glomerular capillary permeability to albumin in isolated glomeruli from Wistar and obese and lean Zucker rats and transmembrane albumin flux in cultured rat podocytes. Expression of PKGIα and upstream proteins was confirmed in the podocytes using Western blotting and immunofluorescence. Insulin (300nM, 5min) increased NAD(P)H-dependent glomerular albumin permeability in Wistar rats and PKGI-dependent transmembrane albumin flux in cultured podocytes. Podocyte exposure to insulin in non-reducing conditions increased PKGIα interprotein disulfide bond formation, altered the phosphorylation of the PKG target proteins MYPT1 and MLC, and disrupted the actin cytoskeleton. The role of NADPH oxidase (NOX) in insulin-induced reactive oxygen species (ROS) generation and insulin-evoked increases in albumin permeability in podocytes was confirmed with NOX2 and NOX4 siRNA. Glomerular albumin permeability was increased in hyperinsulinemic Zucker obese rats with isolated glomeruli showing increased expression of PKGIα and NOX4. Taken together, these data demonstrate that insulin increases glomerular barrier albumin permeability via a PKGI-dependent mechanism involving NAD(P)H-dependent generation of superoxide anion. These findings reveal a role for insulin in the pathophysiology of diabetic glomerular nephropathy.

Involvement of the AMPK–PTEN pathway in insulin resistance induced by high glucose in cultured rat podocytes

As part of the filtration barrier, podocytes play an important role in the development of diabetic nephropathy. Disturbances in insulin signaling accompanied by insulin resistance can lead to various intracellular events. We hypothesized that high glucose concentrations would lead to disturbances in interactions between AMPK and PTEN proteins in podocytes. Experiments were performed in primary rat podocytes cultured with normal (5.6mM) or high (30mM) glucose concentrations for 5d. Immunodetection methods were used to detect AMPK, PTEN, insulin receptor, and Akt proteins, and their phosphorylated forms. Insulin-stimulated changes in glucose uptake were used to detect insulin resistance. Isoforms of AMPK were detected by RT-PCR. AMPK and PTEN activities were modified by metformin, Compound C, siRNA for AMPK isoforms α1 and α2 and siRNA for PTEN, respectively. We found that impairment of insulin induction of glucose uptake into podocytes cultivated in the presence of high glucose concentrations for long periods of time is associated with increased PTEN levels in an AMPK-dependent manner.

Hydrogen peroxide induces activation of insulin signaling pathway via AMP-dependent kinase in podocytes

Podocytes are cells that form the glomerular filtration barrier in the kidney. Insulin signaling in podocytes is critical for normal kidney function. Insulin signaling is regulated by oxidative stress and intracellular energy levels. We cultured rat podocytes to investigate the effects of hydrogen peroxide (H(2)O(2)) on the phosphorylation of proximal and distal elements of insulin signaling. We also investigated H(2)O(2)-induced intracellular changes in the distribution of protein kinase B (Akt). Western blots showed that H(2)O(2) (100 μM) induced rapid, transient phosphorylation of the insulin receptor (IR), the IR substrate-1 (IRS1), and Akt with peak activities at 5 min (Δ 183%, P<0.05), 3 min (Δ 414%, P<0.05), and 10 min (Δ 35%, P<0.05), respectively. Immunostaining cells with an Akt-specific antibody showed increased intensity at the plasma membrane after treatment with H(2)O(2)>. Furthermore, H(2)O(2) inhibited phosphorylation of the phosphatase and tensin homologue (PTEN; peak activity at 10 min; Δ -32%, P<0.05) and stimulated phosphorylation of the AMP-dependent kinase alpha subunit (AMPKα; 78% at 3 min and 244% at 10 min). The stimulation of AMPK was abolished with an AMPK inhibitor, Compound C (100 μM, 2h). Moreover, Compound C significantly reduced the effect of H(2)O(2) on IR phosphorylation by about 40% (from 2.07 ± 0.28 to 1.28 ± 0.12, P<0.05). In addition, H(2)O(2) increased glucose uptake in podocytes (from 0.88 ± 0.04 to 1.29 ± 0.12 nmol/min/mg protein, P<0.05), and this effect was attenuated by Compound C. Our results suggested that H(2)O(2) activated the insulin signaling pathway and glucose uptake via AMPK in cultured rat podocytes. This signaling may play a potential role in the prevention of insulin resistance under conditions associated with oxidative stress.

Extracellular ATP through P2 receptors activates AMP-activated protein kinase and suppresses superoxide generation in cultured mouse podocytes

Podocytes are an important constituent of the glomerular filtration barrier. The function of these glomerular cells is affected by extracellular nucleotides through P2 receptors. The activation of P2 receptors may lead to the activation of NAD(P)H oxidase, the key enzyme in oxidative stress, with the intracellular pathways leading to intracellular ATP depletion associated with an increase in the intracellular AMP:ATP ratio. This deregulation of the energy balance activates AMP-activated protein kinase (AMPK) to restore energy homeostasis. We investigated whether P2 receptor activation influences NAD(P)H oxidase-dependent rate of superoxide anion (O(2)(•-)) generation and AMPK activity in cultured mouse podocytes. The rate of O(2)(•-) generation was measured by chemiluminescence and changes in AMPK activity were determined by immunoblotting against AMPKα-Thr(172)-P. The addition of 100 μM ATP induced a rapid and transient decrease in rate of O(2)(•-) generation and increased AMPK phosphorylation with maximal effects in the first minute (2.44±0.09 versus 1.62±0.06 nmol/mg protein/min, P<0.05 and 0.64±0.04 versus 0.97±0.07, P<0.05, respectively). Both parameters returned to control levels at 10 min. Suramin (300 μM, P2 receptor antagonist) and compound C (100μM, AMPK inhibitor) completely, and STO-609 (25 μM, CaMKK-β inhibitor) partially, prevented ATP action in rate of O(2)(•-) generation and AMPK phosphorylation. Various ATP analogues (10 μM) mimicked the effects of ATP on rate of O(2)(•-) generation and AMPK phosphorylation. The data indicate that extracellular ATP, acting through P2 receptors upstream of CaMKK-β, modulates podocyte function through simultaneous effects on AMPK and NAD(P)H oxidase activities. This mechanism may play a role in restoring energy homeostasis after oxidative stress.

High glucose increases glomerular filtration barrier permeability by activating protein kinase G type Iα subunits in a Nox4-dependent manner

Hyperglycemia is a primary factor that disturbs podocyte function in the glomerular filtration process; this disturbance leads to the development of diabetic nephropathy, and ultimately, renal failure. Podocyte function may also be altered by biological agents that modify protein kinase activity, including the cGMP-activated protein kinase type Iα (PKGIα). We hypothesized that hyperglycemia-induced podocyte protein hyperpermeability was dependent on PKGIα activation, and that PKGIα was activated via dimerization induced by reactive oxygen species. This hypothesis was investigated in rat podocytes cultured in high glucose (HG, 30 mM). Protein expression was measured with Western blot and immunofluorescence. Podocyte permeability was measured with a transmembrane albumin flux assay. We found that HG increased podocyte permeability in long-term incubations (1, 3, and 5 days); permeability was increased by 66% on day 5. This effect was abolished with apocynin, a NAD(P)H inhibitor, and Rp-8-Br-cGMPS, a PKG inhibitor. It was also abolished by introducing small interfering RNAs (siRNAs) against Nox4 and PKGIα into cultured podocytes. Furthermore, HG increased PKGIα dimerization by 138% (0.23 ± 0.04 vs. 0.54 ± 0.09; P<0.05); this effect was abolished with a siRNA against Nox4. Our observations suggested that HG could increase albumin permeability across the podocyte filtration barrier via Nox4-dependent PKGIα dimerization.

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 Ca²⁺-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 β 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.

Expression of GFAT1 and OGT in podocytes: Transport of glucosamine and the implications for glucose uptake into these cells

Glutamine:fructose‐6‐phosphate amidotransferase (GFAT) and N‐acetylglucosaminyltransferase (OGT) participate in glucosamine (GlcN) production and its utilization in O‐glycosylation, one of key post‐translational modifications of nuclear and cytoplasmic proteins. For this purpose, cells require a high rate of intracellular production of GlcN and/or significant GlcN delivery. We studied the expression of GFAT1 and OGT and measured uptake of glucose and GlcN in cultured rat podocytes, the main cellular component of glomerular filtration barrier. RT‐PCR revealed the presence of both GFAT1 and OGT mRNA. Immunofluorescence of GFAT1 has shown staining signal diffused within the cytoplasm of the cell body and processes. However, OGT was distinctly visible around the nucleus and, in diffuse form, within the cytoplasm of cell bodies and processes. Glucose was transported (1.3 ± 0.2 nmol/min/mg protein) mainly by facilitative transporter systems whilst GlcN uptake (1.1 ± 0.2 nmol/min/mg protein) in a significant part, involved a sodium‐dependent transporter. There was interplay between glucose and GlcN uptake. In the presence of GlcN (50 µM), the rate of glucose uptake decreased by about 50%. The rate of GlcN uptake decreased by 28% in the presence of 5.6 mM glucose. Our results suggest that cultured podocytes possess limited ability to synthesize GlcN internally and therefore may need to receive GlcN from the extracellular environment. J. Cell. Physiol. 225: 577–584, 2010.