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.

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 impact of type III sodium-dependent phosphate transporters (Pit 1 and Pit 2) on podocyte and kidney function.

The sodium-dependent phosphate transporters Pit 1 and Pit 2 belong to the solute carrier 20 (SLC20) family of membrane proteins. They are ubiquitously distributed in the human body. Their crucial function is the intracellular transport of inorganic phosphate (Pi) in the form of H2 PO4- . They are one of the main elements in maintaining physiological phosphate homeostasis. Recent data have emerged that indicate novel roles of Pit 1 and Pit 2 proteins besides the well-known function of Pi transporters. These membrane proteins are believed to be precise phosphate sensors that mediate Pi-dependent intracellular signaling. They are also involved in insulin signaling and influence cellular insulin sensitivity. In diseases that are associated with hyperphosphatemia, such as diabetes and chronic kidney disease (CKD), disturbances in the function of Pit 1 and Pit 2 are observed. Phosphate transporters from the SLC20 family participate in the calcification of soft tissues, mainly blood vessels, during the course of CKD. The glomerulus and podocytes therein can also be a target of pathological calcification that damages these structures. A few studies have demonstrated the development of Pi-dependent podocyte injury that is mediated by Pit 1 and Pit 2. This paper discusses the role of Pit 1 and Pit 2 proteins in podocyte function, mainly in the context of the development of pathological calcification that disrupts permeability of the renal filtration barrier. We also describe the mechanisms that may contribute to podocyte damage by Pit 1 and Pit 2.

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.

Lactate regulates respiratory efficiency and mitochondrial dynamics in primary rat podocytes.

Podocytes are crucial for regulating glomerular permeability. They have foot processes that are integral to the renal filtration barrier. Understanding their energy metabolism could shed light on the pathogenesis of filtration barrier injury. Lactate has been increasingly recognized as more than a waste product and has emerged as a significant metabolic fuel and reserve. The recent identification of lactate transporters in podocytes, the expression of which is modulated by glucose levels and lactate, highlights lactate's relevance. The present study investigated the impact of lactate on podocyte respiratory efficiency and mitochondrial dynamics. We confirmed lactate oxidation in podocytes, suggesting its role in cellular energy production. Under conditions of glucose deprivation or lactate supplementation, a significant shift was seen toward oxidative phosphorylation, reflected by an increase in the oxygen consumption rate/extracellular acidification rate ratio. Notably, lactate dehydrogenase A (LDHA) and lactate dehydrogenase B (LDHB) isoforms, which are involved in lactate conversion to pyruvate, were detected in podocytes for the first time. The presence of lactate led to higher intracellular pyruvate levels, greater LDH activity, and higher LDHB expression. Furthermore, lactate exposure increased mitochondrial DNA-to-nuclear DNA ratios and resulted in upregulation of the mitochondrial biogenesis markers peroxisome proliferator-activated receptor coactivator-1α and transcription factor A mitochondrial, regardless of glucose availability. Changes in mitochondrial size and shape were observed in lactate-exposed podocytes. These findings suggest that lactate is a pivotal energy source for podocytes, especially during energy fluctuations. Understanding lactate's role in podocyte metabolism could offer insights into renal function and pathologies that involve podocyte injury.

ADAM10 as a major activator of reactive oxygen species production and klotho shedding in podocytes under diabetic conditions.

Early stages of diabetes are characterized by elevations of insulin and glucose concentrations. Both factors stimulate reactive oxygen species (ROS) production, leading to impairments in podocyte function and disruption of the glomerular filtration barrier. Podocytes were recently shown to be an important source of αKlotho (αKL) expression. Low blood Klotho concentrations are also associated with an increase in albuminuria, especially in patients with diabetes. We investigated whether ADAM10, which is known to cleave αKL, is activated in glomeruli and podocytes under diabetic conditions and the potential mechanisms by which ADAM10 mediates ROS production and disturbances of the glomerular filtration barrier. In cultured human podocytes, high glucose increased ADAM10 expression, shedding, and activity, NADPH oxidase activity, ROS production, and albumin permeability. These effects of glucose were inhibited when cells were pretreated with an ADAM10 inhibitor or transfected with short-hairpin ADAM10 (shADAM10) or after the addition soluble Klotho. We also observed increases in ADAM10 activity, NOX4 expression, NADPH oxidase activity, and ROS production in αKL-depleted podocytes. This was accompanied by an increase in albumin permeability in shKL-expressing podocytes. The protein expression and activity of ADAM10 also increased in isolated glomeruli and urine samples from diabetic rats. Altogether, these results reveal a new mechanism by which hyperglycemia in diabetes increases albumin permeability through ADAM10 activation and an increase in oxidative stress via NOX4 enzyme activation. Moreover, αKlotho downregulates ADAM10 activity and supports redox balance, consequently protecting the slit diaphragm of podocyteσ under hyperglycemic conditions.

Inhibition of phosphodiesterase 5A by tadalafil improves SIRT1 expression and activity in insulin-resistant podocytes.

A decrease in intracellular levels of 3',5'-cyclic guanosine monophosphate (cGMP) has been implicated in the progression of diabetic nephropathy. Hyperglycemia significantly inhibits cGMP-dependent pathway activity in the kidney, leading to glomerular damage and proteinuria. The enhancement of activity of this pathway that is associated with an elevation of cGMP levels may be achieved by inhibition of the cGMP specific phosphodiesterase 5A (PDE5A) using selective inhibitors, such as tadalafil. Hyperglycemia decreased the insulin responsiveness of podocytes and impaired podocyte function. These effects were associated with lower protein amounts and activity of the protein deacetylase sirtuin 1 (SIRT1) and a decrease in the phosphorylation of adenosine monophosphate-dependent protein kinase (AMPK). We found that PDE5A protein levels increased in hyperglycemia, and PDE5A downregulation improved the insulin responsiveness of podocytes with reestablished SIRT1 expression and activity. PDE5A inhibitors potentiate nitric oxide (NO)/cGMP signaling, and NO modulates the activity and expression of SIRT1. Therefore, we investigated the effects of tadalafil on SIRT1 and AMPK in the context of improving the insulin sensitivity in podocytes and podocyte function in hyperglycemia. Our study revealed that tadalafil restored SIRT1 expression and activity and activated AMPK by increasing its phosphorylation. Tadalafil also restored stimulating effect of insulin on glucose transport in podocytes with high glucose-induced insulin resistance. Additionally, tadalafil improved the function of podocytes that were exposed to high glucose concentrations. Our results display novel mechanisms involved in the pathogenesis of glomerulopathies in diabetes, which may contribute to the development of more effective treatment strategies for diabetic nephropathy.

Enhancement of cGMP-dependent pathway activity ameliorates hyperglycemia-induced decrease in SIRT1-AMPK activity in podocytes: Impact on glucose uptake and podocyte function.

Hyperglycemia significantly decreases 3',5'-cyclic guanosine monophosphate (cGMP)-dependent pathway activity in the kidney. A well-characterized downstream signaling effector of cGMP is cGMP-dependent protein kinase G (PKG), exerting a wide range of downstream effects, including vasodilation and vascular smooth muscle cells relaxation. In podocytes that are exposed to high glucose concentrations, crosstalk between the protein deacetylase sirtuin 1 (SIRT1) and adenosine monophosphate-dependent protein kinase (AMPK) decreased, attenuating insulin responsiveness and impairing podocyte function. The present study examined the effect of enhancing cGMP-dependent pathway activity on SIRT1-AMPK crosstalk in podocytes under hyperglycemic conditions. We found that enhancing cGMP-dependent pathway activity using a cGMP analog was associated with increases in SIRT1 protein levels and activity, with a concomitant increase in the degree of AMPK phosphorylation. The beneficial effects of enhancing cGMP-dependent pathway activity on SIRT1-AMPK crosstalk also included improvements in podocyte function. Based on our findings, we postulate an important role for SIRT1-AMPK crosstalk in the regulation of albumin permeability in hyperglycemia that is strongly associated with activity of the cGMP-dependent pathway.

PTEN-induced kinase 1 deficiency alters albumin permeability and insulin signaling in podocytes.

Alterations of insulin signaling in diabetes are associated with podocyte injury, proteinuria, and renal failure. Insulin stimulates glucose transport to cells and regulates other intracellular processes that are linked to cellular bioenergetics, such as autophagy, gluconeogenesis, fatty acid metabolism, and mitochondrial homeostasis. The dysfunction of mitochondrial dynamics, including mitochondrial fusion, fission, and mitophagy, has been observed in high glucose-treated podocytes and renal cells from patients with diabetes. Previous studies showed that prolonged hyperglycemia is associated with the development of insulin resistance in podocytes, and high glucose-treated podocytes exhibit an increase in mitochondrial fission and decrease in markers of mitophagy. In the present study, we found that deficiency of the main mitophagy protein PTEN-induced kinase 1 (PINK1) significantly increased albumin permeability and hampered glucose uptake to podocytes. We suggest that PINK1 inhibition impairs the insulin signaling pathway, in which lower levels of phosphorylated Akt and membrane fractions of the insulin receptor and glucose transporter-4 were observed. Moreover, PINK1-depleted podocytes exhibited lower podocin and nephrin expression, thus identifying a potential mechanism whereby albumin leakage increases under hyperglycemic conditions when mitophagy is inhibited. In conclusion, we found that PINK1 plays an essential role in insulin signaling and the maintenance of proper permeability in podocytes. Therefore, PINK1 may be a potential therapeutic target for the treatment or prevention of diabetic nephropathy.

Cathepsin C is a novel mediator of podocyte and renal injury induced by hyperglycemia.

A growing body of evidence suggests a role of proteolytic enzymes in the development of diabetic nephropathy. Cathepsin C (CatC) is a well-known regulator of inflammatory responses, but its involvement in podocyte and renal injury remains obscure. We used Zucker rats, a genetic model of metabolic syndrome and insulin resistance, to determine the presence, quantity, and activity of CatC in the urine. In addition to the animal study, we used two cellular models, immortalized human podocytes and primary rat podocytes, to determine mRNA and protein expression levels via RT-PCR, Western blot, and confocal microscopy, and to evaluate CatC activity. The role of CatC was analyzed in CatC-depleted podocytes using siRNA and glycolytic flux parameters were obtained from extracellular acidification rate (ECAR) measurements. In functional analyses, podocyte and glomerular permeability to albumin was determined. We found that podocytes express and secrete CatC, and a hyperglycemic environment increases CatC levels and activity. Both high glucose and non-specific activator of CatC phorbol 12-myristate 13-acetate (PMA) diminished nephrin, cofilin, and GLUT4 levels and induced cytoskeletal rearrangements, increasing albumin permeability in podocytes. These negative effects were completely reversed in CatC-depleted podocytes. Moreover, PMA, but not high glucose, increased glycolytic flux in podocytes. Finally, we demonstrated that CatC expression and activity are increased in the urine of diabetic Zucker rats. We propose a novel mechanism of podocyte injury in diabetes, providing deeper insight into the role of CatC in podocyte biology.

Application of local innervated skin flaps in healing of posterior and sole calcanea surfaces defects.

In the paper were shown the methods of the local innervated skin flaps, harvested from the posterior surface of the lower leg, the lateral malleolus region and the plantar surface of the foot, used in treatment of soft tissues defects of posterior and plantar surface of the heel.

[Experimental and clinical of the range of the posterior tibial island flap]. / Badanie doswiadczalne i kliniczne zasiegu wyspowego plata piszczelowego tylnego.

Basing on anatomical studies (20 dissected lower extremities) and clinical experiences (4 cases), the paper presents local transposition possibilities of a distally pedicled tibial flap (pedicled on the posterior tibial artery). The flap allowed for soft-tissue coverage of 2/3 of the plantar side of the foot and 1/4 of the dorsal aspect of the foot.