FP230
ROLE OF PROTEASE-ACTIVATED RECEPTORS IN REGULATION OF CALCIUM SIGNALING IN PODOCYTES IN TYPE 2 DIABETIC NEPHROPATHY

INTRODUCTION: Protease-activated receptors (PARs) can be involved in glomerular, microvascular and inflammatory regulation of renal function under both normal and pathological conditions. The elevation of concentrations of serine proteases activating PARs in plasma and urine is associated with the development of albuminuria and proteinuria in both patients and experimental rodent models. However, the direct cell-specific mechanisms as well as type of serine proteases and PARs involved in the development of diabetic kidney disease (DKD) are not well defined.

METHODS: The type 2 diabetic nephropathy (T2DN) rat model strain was developed at Medical College of Wisconsin by crossing diabetic Goto-Kakizaki (GK) and FHH (Fawn Hooded Hypertensive) rats.  T2DN rats were used here to study contribution PAR-mediated signaling towards the development of DKD.  GK and Wistar rats were selected as diabetic and  non-diabetic controls, respectively.  Using our established technique of isolation and confocal ratiometric imaging of intracellular calcium dynamics in glomeruli podocytes, we explored the involvement of PARs and serine proteases in DKD.  Thrombin, kallikrein or urokinase, as well as corresponding PAR-activating small synthetic peptides (TFLLR-NH2 (TRAP6), AY-NH2 and SLIGRL-NH2) were applied to test changes in intracellular calcium [Ca2+]i concentrations in glomeruli podocytes.

RESULTS: In our initial study we characterized progression of DKD in T2DN male and female rats.  Renal structural abnormalities such as glomerular and tubular hypertrophy were observed at early ages (12 weeks) and precede the development of proteinuria.  By 9-12 months of age T2DN rats exhibit significantly elevated, compared to GK and Wsitar rats, proteinuria, accompanied by renal histologic abnormalities such as FSGS, mesangial matrix expansion, and thickening of basement membranes.  Female T2DN rats were protective with regards to the developments of diabetes and kidney injury compared to male rats of the same age.  The development of DN was also associated with enhanced urinary urokinase levels in T2DN male rats.  Scoring of glomerular damage also revealed aggravated glomerular injury in T2DN rats.  Basal [Ca2+]i level in podocytes was within normal range in Wistar and GK rats, whereas in T2DN rats it was pathologically elevated already at 12 weeks age.  IHC and Western blot analyses revealed that PAR1 and PAR4 expression was significantly elevated in T2DN rats and human kidneys from diabetic patients.  Furthermore, T2DN rats have a significant elevation in calcium signaling in response to tested serine proteases compared to control strains.  Single channel analysis (the cell-attached configuration of the patch clamp method) was further used to assess TRPC channels activity in the podocytes in freshly isolated glomeruli.  Application of thrombin (10 μM) resulted in acute activation of endogenous TRPC channels, which demontrate contribution of these channels in PAR-mediated signaling.

CONCLUSIONS: Collectively, these data implicate the direct involvement of serine proteases in the modulation of [Ca2+]i signaling, which might lead to podocytes injury, apoptosis and the development of nephrotic syndrome in diabetes. Therefore, PARs and corresponding serine proteases become a new potential therapeutic target for the prevention of DKD.