LA-induced mitochondrial Ca²⁺ efflux was responsible for peroxynitrite generation.

<p>Mitochondria were prepared from HM cells and labeled with X-rhod-1 (2 µM) and DCF (1 µM) with (A) or without Ca²⁺ (B). LA-induced mitochondrial Ca²⁺ efflux (black traces) and peroxynitrite generation (red traces) were indexed by the gradual decrease in X-rhod-1 and increase in DCF fluorescence intensity, respectively.</p

LA-induced mitochondrial Ca²⁺ efflux and protein nitrotyrosylation were enhanced in the kidney of db/db mice.

<p>Mitochondria were prepared from fresh kidney tissues of 12–16 wk old db/+ and db/db mice and labeled with X-rhod-1 (2 µM). (A): LA-induced mitochondrial Ca²⁺ efflux in the kidney of db/+ and db/db mice. (B): The ratio of the initial rate of LA-induced mitochondrial Ca²⁺ efflux (V_LA; measured in the first 60 s following LA) to the basal rate (V₀; measured prior to LA). The values represent mean±SEM of V_LA/V₀, **<i>P</i><0.01, n = 3. (C) and (D): Nitrotyrosine levels in the kidney homogenates of four pairs of db/+ and db/db mice were analyzed by western blot using α-tubulin as loading control. The arrowhead highlights a protein band (∼22 kDa) with enhanced nitrotyrosine levels in db/db mice, **<i>P</i><0.01, n = 4. (E) and (F): Representatives of the nitrotyrosine levels in the kidney sections of db/+ and db/db mice as determined by immunohistochemistry.</p

Mitochondrial complexes I and III were required for LA-induced peroxynitrite generation.

<p>HM cells were labeled with fura-2 and DCF to measure LA responsive [Ca²⁺]_i mobilization (black traces) and peroxynitrite generation (red traces). (A): LA responses in untreated cells (control); (B) LA responses in cells pretreated with rotenone (a mitochondrial complex I inhibitor, 10 µM, 20 min); (C) and (D): LA responses in cells pretreated with the mitochondrial complex III blockers, antimycin A (1 µM, 20 min; C) or myxothiazol (0.5 µM, 60 min; D).</p

Mechanism of excessive NEFA contributing to pathogenesis of diabetic nephropathy.

<p>NEFA, such as linoleic acid (LA, an 18∶2 n-6 polyunsaturated fatty acid) induces Ca²⁺ efflux from mitochondria by activating a hsp90β1-dependent pathway (PIMCE). The LA responsive mitochondrial Ca²⁺ efflux diminishes the inhibitory effect of Ca²⁺ on superoxide production from complexes I or III and enhances nitrite conversion to NO by complex III, resulting in increased peroxynitrite formation and protein nitrotyrosylation. Protein nitrotyrosylation may disrupt the normal functions of mesangial cells and the kidney, leading to abnormalities in the structure and/or function of diabetic kidney.</p

The effect of hsp90β1 on LA-induced [Ca²⁺]_i mobilization and peroxynitrite generation.

<p>HM cells were treated with vehicle (control), hsp90β1 RNAi, or 17-DMAG for 48 h. (A): Cell lysates were prepared and the alterations of hsp90β1 expression were analyzed by western blot analysis using α-tubulin as loading control. (B): The alteration of LA-induced [Ca²⁺]_i mobilization and peroxynitrite generation in hsp90β1 RNAi- and 17-DMAG-treated HM cells relative to control. The values represent the mean±SEM. *<i>P</i><0.05, **<i>P</i><0.01, n = 6.</p

Mitochondria played an essential role in LA-induced [Ca²⁺]_i mobilization and peroxynitrite generation.

<p>HM cells were labeled with fura-2 and DCF to measure [Ca²⁺]_i mobilization (black traces) and peroxynitrite generation (red traces). (A): Bradykinin (BK, 100 nM) responses; (B): thapsigargin (TG, 2 µM) responses; (C) and (F): LA responses measured in the presence of extracellular Ca²⁺ (control); (D): LA responses measured without extracellular Ca²⁺; (G) LA responses measured in cells pretreated with BAPTA (50 µM, 30 min) without extracellular Ca²⁺. In the absence of extracellular Ca²⁺, (I): LA responses (control); (J): LA responses in cells pretreated with TG (2 µM, 5 min); (K) LA responses in cells pretreated with FCCP (a mitochondrial uncoupler; 4 µM, 5 min). The values in graphs (E, H, and L) represent the mean±SEM of the relative amplitudes of LA-induced [Ca²⁺]_i and peroxynitrite responses, *<i>P</i><0.05, **<i>P</i><0.01, n = 6, treated cells <i>vs</i> control.</p

Interaction of a Hydrophobic-Functionalized PAMAM Dendrimer with Bovine Serum Albumin: Thermodynamic and Structural Changes

The interaction between a hydrophobic-functionalized PAMAM dendrimer (PAMAM-NH₂-C12, 25%, G4) and bovine serum albumin (BSA) has been investigated by circular dichroism (CD), UV–vis, and fluorescence spectroscopic methods and molecular modeling. The analysis of the effects of dendrimer complexation on the stability and conformation of BSA indicated that the binding process of the hydrophobic-functionalized dendrimer with BSA induced the relatively large changes in secondary structure of protein. Thermal denaturation of BSA, when carried out in the presence of dendrimer, also indicated that this hydrophobic-functionalized dendrimer acted as a structure destabilizer for BSA. The hydrophobic, electrostatic, and hydrogen bonding forces played important roles in the complex formation. The putative binding site of PAMAM-NH₂-C12 (25%) dendrimer on BSA was near to domain I and domain II. The effect of hydrophobic modification on the stability and structure of BSA would find useful information on the cytotoxicity of PAMAM dendrimer

Impact of 17-DMAG on hsp90β1, [Ca²⁺]_m, and peroxynitrite generation in the kidney.

<p>Western blot analysis was performed to assess hsp90β1 in kidney homogenate (<i>A</i>) and isolated mitochondria (<i>B</i>) of HFD-fed <i>db/db</i> mice with 6 animals per group. Linoleic acid (LA)-induced [Ca²⁺]_m efflux and peroxynitrite generation (<i>C–F</i>) in kidney mitochondria were measured.</p

Kidney histopathology of HFD-fed <i>db/db</i> mice.

<p>Representative figures showing the photomicrographs of HE (<i>A–F</i>), PAS (<i>G</i>, <i>H</i>), and Masson's trichrome stained sections from <i>db/db-HF-S</i> group (<i>C, E, G, I</i>) and <i>db/db-HF-G</i> group (<i>D, F, H, J</i>) taken at 200× magnification. Image-based computer assisted analysis was performed to quantify tubular damage index (<i>K</i>), mesangial expansion (<i>L</i>), and interstitial collagen accumulation (<i>M</i>) from 6 animals per group. (A) and (B) showed parallel experiments with HFD-fed <i>db/+</i> mice injected with saline and 17-DMAG, respectively.</p

Effect of 17-DMAG on survival rate of HFD-fed <i>db/db</i> mice.

<p>Kaplan-Meyer survival analysis was performed using the log-rank statistics to measure the difference between the survival curves of d<i>b/db-HF-S</i> vs <i>db/db-HF-G</i> mice with n = 9 per group. Parallel experiments were performed with <i>db/+</i> mice (<i>db/+-HF-S</i> and <i>db/+-HF-G</i> groups) and no mortality was observed.</p