Bioactive Peptides Control Receptor for Advanced Glycated End Product-Induced Elevation of Kidney Insulin Receptor Substrate 2 and Reduce Albuminuria in Diabetic Mice

Despite the availability of agents for controlling blood sugar, satisfactory glycemic control continues to be a challenge for the rapidly expanding diabetic population of industrialized nations and is associated with both macrovascular and microvascular complications of type 2 diabetes [1, 2]. Kidney failure is one economically relevant consequence resulting from sustained elevations in blood sugar. According to the American Diabetes Association, diabetes is the leading cause of kidney failure, accounting for about 40% of new cases. Interventions that would delay the progression of diabetic nephropathy in patients with poorly controlled blood sugar could have a significant impact on healthcare costs.In diabetic humans and db/db mice, the receptor for advanced glycated end products (RAGE) is activated by systemic ligands such as amphoterin and glycated hemoglobin [3]. RAGE has been implicated in the development of kidney dysfunction consequent to elevated blood sugar [4]. The intracellular biochemical events downstream of RAGE activation leading to the loss of kidney function and albuminuria in db/db mice are not well understood. RAGE blockade through the use of soluble RAGE decoys has been proposed as a method for controlling complications of diabetes in humans [5, 6].Kidney mesangial cell matrix expansion characterized by excessive deposition of collagen IV and fibronectin is an often cited correlate of disease progression [7]. However, effective interventions based on this hypothesis have yet to be developed. Recently, the inhibition of protein kinase C (PKC) isoforms has been proposed as a possible therapeutic intervention for kidney disease [8]. A peptide capable of inhibiting PKC-β2 in cultured cells has been described [9].Correlation matrices or dendograms [10] constructed from RAGE-adaptive datasets gathered in cultured kidney cell and kidney tissue extracts can help identify reliable biochemical correlates of disease and can guide the development of effective therapeutic interventions. Although correlations do not reveal causative links, the clustering of biochemical correlates can help define ‘virtual dysregulations’ around which hypothesis-driven interventions can be designed and tested.Humanin is a 24-amino-acid natural human peptide recently identified in the context of neurodegenerative disease [11]. Substitution mutants and fusions of humanin with other sequences can generate molecules with altered properties in vivo [12]. The underlying mechanism for proposed anti-apoptotic effects of humanin is not well understood.We have previously described a short metal-binding domain (MBD) of insulin-like growth factor-binding protein 3 with cell targeting and cell internalization properties. Fusion of other peptide or protein sequences with the MBD generates molecules that are efficiently delivered into cells [13].In this study, we have surveyed a panel of intracellular biochemical readouts in cultured 293 kidney cells challenged with glycated hemoglobin and various chemical and peptide inhibitors. From these data we have selected a subset of readouts that are significantly impacted by RAGE ligand in these cells. Kidney extracts from albuminuric db/db mice were assayed for these selected biochemical markers. Correlation matrices constructed from these data suggest possible modifications to our current understanding of diabetic kidney disease.Humanin (wild type) and S14G-humanin were purchased from American Peptide Co., Sunnyvale, Calif., USA. NPKC (AKKGFYKKKQCRPSKGRKRGFCWPSIQITSLNPEWNET) and P38 (AKKGFYKKKQCRPSKGRKRGFCWAPSRKPALRVIIPQAGK) peptides containing the MBD domain of insulin-like growth factor-binding protein 3, which provides effective biodistribution, cell internalization and nuclear delivery for linked sequences [13], were synthesized and purified by Genenmed Synthesis, Inc., San Francisco, Calif., USA. Glycated hemoglobin, amphoterin, tumor necrosis factor-α, epidermal growth factor (EGF), resistin, insulin, SDKP, caffeine, rapamycin and the antibodies anti-insulin receptor substrate 1 (IRS-1), anti-RAGE, anti-fibronectin, anti-IRS-1 (Ser307) and anti-IRS-2 (Ser731) were purchased from Sigma Chemical Co., St. Louis, Mo., USA. The following reagents were obtained from EMD Chemicals, San Diego, Calif., USA: AKT (Ser473)-blocking peptide, AKT inhibitors (II–IX), JNK inhibitors II and III, SB203580, LY294002 and PD98059. PhosphoSafe tissue cell extract reagent was from Novagen, Madison, Wisc., USA. Cell culture reagents RPMI-1649, DMEM and FBS were from Hyclone, Logan, Utah, USA. Protein concentration kit was purchased from Pierce Biotechnology, Rockford, Ill., USA. Antibodies to the following antigens were purchased from the indicated suppliers: c-Jun (Ser63), c-Jun (Ser73), c-myc (Ser62) and c-myc (Thr58) from EMD Chemicals; Erk1/2 (Thr202/Tyr204), P38 mitogen-activated protein kinase (MAPK; T180/Y182), SAPK/JNK (Thr183/Ty185), P38-α/SAPK2a, c-myc (Thr58Ser52), PKC-β2, phospho-PKC-α/β2 (Thr638/641), PKC-δ, PKC-δ/θ, PKC-θ, PKC-ζ/λ, PKD/PKC-µ (Ser916), PKD/PKC-µ (Ser744/748), PKD/PKC-µ, AKT (Thr308), AKT (Ser473), AKT1, AKT2, AKT3, MKK3/MKK6 (Ser189/207), activating transcription factor 2 (ATF2; Thr71), paxillin (Y118) and gsk3B (Ser9) from Cell Signaling, Danvers, Mass., USA; collagen IV and IRS-2 from RnD Systems, Minneapolis, Minn., USA.Cells were passaged in DMEM plus 10% FBS and plated in 6-well plates. When 90–95% confluent, they were treated with different reagents for 4 h. Cells were collected and washed twice with 1× PBS. Extracts were made in 200 µl PhosphoSafe buffer and diluted in 1× PBS to set up ELISAs.Human kidney mesangial cells and media were purchased from Lonza, Walkersville, Md., USA. Cells grown in mesangial cell basal media that were quiescent for 2 days were treated with glycosylated hemoglobin and peptides, and cell extracts were prepared and assayed by ELISA in exactly the same manner as described for 293 cells.db/db mice were purchased from Jackson Laboratories. Animals with blood glucose <200 mg/dl in week 8 were sacrificed and used as null controls. Remaining animals were randomized into 4–8 animals per treatment group and were injected by subcutaneous bolus daily from week 8 through 13 (first experiment) or week 9–15 (second experiment). At the beginning and end of each experiment, each mouse was housed in an individual metabolic cage for a 24-hour urine collection. The volume of urine collected was recorded. Urine samples were assayed for albumin by ELISA, and the total amount of albumin excreted was calculated by multiplying the volume of urine by the concentration of albumin in the urine. Diabetes progression was monitored weekly during treatment by measuring blood glucose levels. Animals were sacrificed at week 13 (first experiment) or week 15 (second experiment). At termination, plasma and organs (right and left kidneys, pancreas, brain, heart, liver) were collected for preparation of tissue extracts and ELISA assays. Organ slices were ground in cell lysis buffer, and total protein concentration was measured using a bicinchoninic acid protein assay kit.Insulin levels were determined in plasma samples with the Ultrasensitive Mouse Insulin ELISA from Alpco Diagnostics (Windham, N.H., USA). Blood was collected in heparin-coated capillary tubes, and red blood cells were separated by centrifugation at 5,000 rpm for 5 min. Plasma glucose was assessed by pipetting 5-µl samples on glucometer strips and reading in the One Touch Basic Glucometer (LifeScan Canada Ltd., Burnaby, BC, Canada). Mice were fasted overnight prior to the glucose test.Extracts were diluted 1:25, and 100 µl of each sample was added to a 96-well plate. After 1 h, the plate was washed (3 times with 1× PBS + Tween). Three percent BSA was added to the plates and incubated for 1 h. The wash step was repeated, and then primary antibody was added for 1 h. Another wash step was followed by treatment with secondary antibody for 1 h. The wash was again repeated, and 100 µl per well tetramethylbenzidine was added. After incubation for 15 min, the samples were read in a plate reader at 655 nm.Immunoprecipitation was done using the Catch and Release IP Kit (Millipore, Billerica, Mass., USA) according to the manufacturer’s specifications. Briefly, HEK293 cells were treated with either saline, glycated hemoglobin or amphoterin for 4 h. The cells were collected and washed 2 times, and whole-cell extracts were prepared in PhosphoSafe buffer. Three hundred microliters of each extract was mixed with 10 µl anti-phosphatidylinositol 3 (PI3)-kinase antibody for 60 min at 4°C with gentle rocking. The samples were then applied to the column and centrifuged for 30 s. The column was washed 3 times, and then 400 µl of elution buffer was added to the column and centrifuged at 5,000 rpm for 30 s to collect all samples. The purified material was assayed for IRS-2 by ELISA.Probability values (p values) were computed using Student’s t test. Unless otherwise stated, p values are expressed relative to saline-treated controls.Figure 1a shows that HEK293 kidney cells cultured in the presence of RAGE ligands amphoterin and glycated hemoglobin for 4 h exhibit marked and sustained elevations of total cellular IRS-2 (but not IRS-1) and PI3-kinase-associated IRS-2. Fibronectin is significantly elevated only after 7–8 h of treatment, but collagen IV elevation is sustained over several hours and parallels that of IRS-2 (fig. 1b). A preliminary survey of cell extracts by ELISA (31 markers tested, data not shown) revealed an unusual pattern of sustained intracellular phosphorylation events affecting several key molecules including a remarkable and selective phosphorylation of PKB/Akt at Ser473 (but not Thr308), phosphorylation of IRS-1 (Ser312) but not IRS-2 (Ser731), and phosphorylation of PKC-α/β2 (Ser638/641) but not PKC-µ (Ser916). In addition, JNK (Thr183/Tyr185) and the P38 MAPK target ATF2 (Ser71) were selectively phosphorylated, but ERK (Thr202/Tyr204) was not. These data are summarized in table 1. In order to show that this set of RAGE-responsive adaptations in intracellular biochemistry leads to significantly modified responses to the extracellular milieu, we showed dramatically altered phosphorylation of key residues Thr308 and Ser473 in Akt in response to a range of growth, metabolic and inflammatory signals in cells that had been pretreated with glycated hemoglobin (fig. 2).The influence of selected inhibitors (Akt inhibitor IV, rapamycin and LY290004) and of the bioactive peptides humanin, NPKC and Akt-Ser473-blocking peptide on a selected set of RAGE-activated biochemical events is shown in figure 3. Humanin and NPKC peptides partially reverse the elevations in IRS-2 and Akt1 levels but not the selective phosphorylation of Akt-Ser473. Conversely, the latter can be blocked by Akt-Ser473-blocking peptide, without affecting IRS-2 and Akt1 levels. LY290004, a selective inhibitor of PI3-kinase, and rapamycin, an mTORC1 inhibitor, further elevates IRS-2 and Akt, suggesting that these events are independent of the PI3-kinase pathway and mTORC1. Taken together, the pattern of inhibition and stimulation suggests the presence of 2 regulons, 1 defined by IRS-2 and Akt1 (IRS-2 regulon) and 1 by the selective phosphorylation of Akt-Ser473 and JNK-Thr183/Tyr185 (designated ‘stress regulon’ because of JNK stress kinase). In human kidney mesangial cells pretreated with glycated hemoglobin, IRS-2 levels are significantly reduced by exposure to either S14G-humanin or NPKC peptides (table 2).In order to test the effect of subcutaneously injected peptides in diabetic mice, 8-week-old db/db mice were treated daily for 5 weeks with the indicated subcutaneous bolus doses of humanin or NPKC peptide. Wild-type humanin was compared with the S14G substitution mutant (previously reported by others to be more active), and the wild-type peptide was surprisingly found to be more effective. Figure 4 shows the results obtained from the measurement of (1) physiological markers such as urine albumin excretion, body weight, plasma glucose and insulin, and (2) ELISAs of kidney tissue extracts assayed for the markers defined in the RAGE-inducible set derived from 293 cell culture experiments, as summarized in table 1. Peptide-mediated improvements in albuminuria occurred in the absence of any significant effect on body weight or on the elevated circulatory levels of glucose and insulin. For the purpose of displaying the data, kidney tissue markers are organized into 6 ‘virtual regulons’ defined by pairwise Pearson correlation analysis using ELISA value sets derived from 30 individual animals. The boundaries of each tightly correlated cluster defining a ‘virtual regulon’ are defined arbitrarily. Humanin and NPKC help normalize kidney IRS-2 levels and albuminuria. Humanin additionally influences collagen IV and Akt1 (regulons 3 and 4), as seen in short-term cell culture experiments, but the direction of Akt1 modulation in chronic kidney disease is the opposite of what is observed with short-term treatment of 293 cells. Unlike the observed lack of effect in 293 kidney cell culture, chronic treatment of db/db mice with humanin helps normalize p-Akt-Ser473 and p-JNK-T183/Y185 levels, 2 tightly linked markers in regulon 1 (‘stress regulon’).In order to examine the possibility of an obligate relationship between collagen IV synthesis and albuminuria, 9-week-old db/db mice were treated for 5 weeks with 40 µg/day subcutaneous bolus P38 peptide (an intracellular inhibitor of activated P38 MAPK target ATF2 that includes an MBD domain sequence for cell internalization and nuclear delivery of the peptide in vivo) or humanin peptide. The results (table 3) show a marked reduction in collagen IV in P38 peptide-treated animals, but in these animals, a significant exacerbation of albuminuria is observed. Kidney tissue IRS-2 is also elevated in P38-treated animals relative to saline-treated controls (0.205 ± 0.007 versus 0.184 ± 0.009 arbitrary units; p = 0.028). As in the first experiment, humanin reversed albuminuria.Treatment of db/db mice with bioactive peptides humanin and NPKC ameliorates albuminuria. Kidney tissue extracts were used to generate an adaptive dataset of biochemical markers. Correlation matrices based on these datasets reveal tightly clustered readouts which may, in turn, provide potentially fundamental insights into the adaptive circuitry of kidney cells. Readout clusters may be considered ‘virtual regulons’ for the purpose of guiding the hypothesis-driven design and development of novel and targeted therapeutic approaches to disease. The underlying assumption of this approach is that cellular responses to environmental insults are adaptive (or maladaptive, in the case of disease) and may expose universal aspects of adaptive logic such as characteristic responses to stress [14, 15], enhanced plasticity [16, 17] or increased internality of decision making as revealed, for example, by the temporarily modified response to endocrine and metabolic signals summarized in figure 2.IRS-1 and IRS-2 proteins are central integrators of signaling traffic from cell membrane receptor tyrosine kinases responding to metabolic and growth signals, especially insulin and insulin-like growth factors [18], and may be of particular relevance in diabetes [19]. Although selective action of IRS isoforms has been proposed for specialized settings such as metastasis [20], the existence of a universal cellular logic switch based on the ratio of total active IRS-2 to IRS-1 has not been previously postulated. We show that in cultured 293 kidney cells challenged with glycated hemoglobin, as well as in kidney extracts from diabetic mice, a marked elevation in total IRS-2 – but not IRS-1 – levels is observed, accompanied by higher levels of phosphorylated IRS-1/Ser307, which has been linked to insulin resistance [21], but not of phosphorylated IRS-2/Ser731. These types of changes would be expected to result in an increased involvement of IRS-2 in signaling events through the PI3-kinase pathway leading to activation of PKB/Akt. We show a significantly elevated level of IRS-2 associated with PI3-kinase in cells treated with RAGE ligand.Akt is a central consolidator of cellular logic [22]. Fully activated Akt is phosphorylated at 2 key residues, Thr308 and Ser473. Differential phosphorylation of Akt at these residues has been previously described [23, 24]. RAGE-mediated changes in 293 kidney cells involve altered signaling in the IRS-Akt axis. In db/db mice exhibiting elevated albuminuria, Akt1 levels are coupled to albumin excretion which is, in turn, coupled to Akt/Ser473 (but not Akt/Thr308) phosphorylation. In cultured 293 cells challenged with glycated hemoglobin, similarly linked responses are observed, with differential phosphorylation at Ser473 (inhibited by Ser473-blocking peptide), and consequently, altered responses to insulin and EGF signaling. LY20004, a specific inhibitor of PI3-kinase, enhances the preferential phosphorylation of Ser473, suggesting that the event is independent of the PI3-kinase cascade. Although the rapamycin-insensitive mTOR complex mTORC2, which contains Rictor, has been recently implicated as the elusive PDK2 responsible for the phosphorylation of Akt-Ser473 [23], rapamycin appears to reduce Ser473 phosphorylation in kidney cells. Other enzymes, such as PKC, have also been implicated as potential kinases for Akt-Ser473 [25]. Preferential phosphorylation of Akt-Ser473 in a PI3-kinase-independent manner may be part of the adaptive response characterized by elevated IRS-2 levels.In this work, we have surveyed a panel of intracellular biochemical readouts in cultured 293 kidney cells challenged with glycated hemoglobin and various chemical and peptide inhibitors. As shown in table 2, similar data can be obtained from cultured human kidney mesangial cells. We elected to use 293 cells for most experiments because of better assay reproducibility, ease of culture and handling and lower cost of materials for routine assay use.Treatment of db/db mice with 20 µg/day subcutaneous bolus humanin or 40 µg/day NPKC peptide for 5 weeks ameliorates albuminuria and lowers IRS-2 levels. In addition, humanin helps normalize a cluster of RAGE-mediated biochemical effects, without affecting circulatory levels of glucose or insulin. Similar effects of humanin on biochemical markers can be observed as a result of short-term treatment of cultured kidney cells, except that the modulation of Akt1 is in the reverse direction. Treatment with wild-type humanin is more effective than with the S14G variant, which has been shown to be more active in models of neurodegenerative disease order to further the between albuminuria and the biochemical readouts that may be significantly altered by correlation matrices were from a dataset derived from ELISAs of kidney extracts prepared from 30 db/db In these biochemical readouts cluster into ‘virtual Humanin and NPKC to influence the readouts that correlate most with of using the NPKC peptide ameliorates albuminuria and IRS-2 levels in the of treated However, humanin, NPKC not normalize the elevation in and 2 markers the ‘stress In kidney extracts = and in 293 kidney cell culture = these 2 markers in response to environmental not The of responses to humanin and NPKC with to these markers suggests a between linked to albuminuria and more stress responses by exposure to or treatment of diabetic mice with peptide P38 as an intracellular inhibitor of activated albuminuria inhibiting collagen IV is with the hypothesis that biochemical changes linked to a stress response may not be as linked to albuminuria as are IRS-2 together, our data from kidney extracts and cultured kidney cells suggest that humanin by biochemical most associated with kidney disease as well as associated with a more stress the other NPKC may on a more subset of biochemical IV a of matrix can be from albuminuria in animals treated with P38 the peptide dramatically collagen synthesis but protein albuminuria is tightly linked to plasma glucose and body weight, humanin dramatically ameliorates protein excretion in the urine without any significant impact on plasma glucose and insulin levels or body markers by or stress may be from that have a primary to kidney a between IRS-2 elevation and albuminuria is not by our data, we that the adaptive of cellular IRS-2 levels from of IRS-1 a potentially biochemical correlate of kidney disease in diabetic The human peptide humanin, previously to have a function in neurodegenerative has a effect on IRS-2 elevation both in in and may be a for therapeutic intervention in kidney and for

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