1,Treatment With Peroxisome Proliferator-Activated Receptor-gamma (PPARγ) Agonist Is Protective In Podocyte Injury-Associated FSGS 2,Absence Of Integrin β6 Does Not Change Interstitial Fibrosis But Worsens Glomeruloslerosis In Remnant Kidney Mice

PART 1 Treatment with Peroxisome Proliferator-Activated Receptor-gamma (PPARγ) Agonist Is Protective in Podocyte Injury-Associated FSGSBackground We have previously observed increased expression of PPARγ in podocytes in both rat and human sclerotic conditions in vivo. PPARγ agonist ameliorated non-diabetic glomerulosclerosis (FSGS) in the hypertensive subtotal nephrectomy model in the rat, suggesting that up-regulated podocyte PPARγ in sclerosis could be a beneficial counterregulatory response. Our recent in vitro data show that PPARγ activation protects against puromycin aminonucleoside (PAN)-induced apoptosis and necrosis. The aim of the present study was to investigate whether activation of PPARγ can also attenuate podocyte injury-associated glomerulosclerosis in vivo.Methods PAN nephropathy, a podocyte-injury model of FSGS, was induced in adult male Sprague-Dawley rats. Following uninephrectomy at week 0, i.p. PAN was given at weeks 2 (100mg/kg Bwt), 6, 7 and 8 (40mg/kg Bwt). The animals then either received no further treatment, Control group (Cont); or pioglitazone (10 mg/kg/d, Pio) starting at week 0, P0 group; or pioglitazone starting at week 6, P6 group. Morphological and molecular biological analyses were performed at week 12. Results At week 12, there were similar urinary protein excretion and SBP during three groups. GFR was decreased in CONT and P0 groups by week 12; Rats with posttreatment with Pio had increased GFR, and by morphological analyses, this group showed parallel amelioration of the development of glomerulosclerosis compared with control. PPARγ expression in PAN-treated rats at 12 weeks was present in podocytes and in mesangial cells in segmentally sclerotic glomeruli, with occasional parietal epithelial cell staining. In PAN+Pio rats, mesangial staining was lessened, but podocyte staining was strongly accentuated, correlating with less severe overall sclerosis. Treatment with Pio partially restored podocyte WT-1 staining and showed a numerical trend to decrease the ratio of PCNA-positive to apoptoic cells in glomeruli. Pre- or post-treatment with Pio significantly reduced infiltrating glomerular macrophages and PAI-1 mRNA expression in cortex. In contrast, Pio did not change renal mRNA levels for TGF-β1 and TIMP-1. Pio treatment decreased renal cortical Aglp4 expression to almost 20% at Cont group and increased VEGF positive glomerular cells.Conclusions We conclude that treatment with the PPARγ agonist pioglitazone has protective effects on progression of podocyte injury-associated glomerulosclerosis. These beneficial effects appear to be related to the regulation of podocyte apoptosis, decreased macrophage infiltration and PAI-1 expression, and protection of glomerular capillary loss by increasing VEGF expression and decreasing Aglp4. Our results further indicate that there are key functional and morphological differences in altimate responses to PPARγ agonist given before, or after, podocyte injury, with PPARγ protection only occurring in the latter. The beneficial affects of delayed time point of interaction is clinically relevout, as therapies are initiated in humans following the initial insult. Our results thus suggest that PPARγ agonists can provide a novel approach with therapeutic potential in podocyte injury-associated FSGS.PART2Absence of integrin αvβ6 does not change interstitial fibrosis but worsens glomeruloslerosis in remnant kidney miceBackground The heterodimeric integrin αvβ6 activates transforming growth factor-β (TGFβ) locally. Integrin αvβ6 is expressed in the tubular epithelium and the juxtaglomerular apparatus (JGA) in kidney. We have previously observed that mice deficient in β6 (β6-/-) were protected against interstitial fibrosis induced by unilateral ureteral obstruction. The aim of the present study was to investigate whether the effect of β6 deficiency on development of glomerulosclerosis was parallel to that on interstitial fibrosis, by study of the remnant kidney model of glomerulosclerosis and associated, secondary interstitial fibrosis.Methods Adult male wild type (WT 129) and β6-/- mice on 129 background underwent 5/6 nephrectomy (Nx). Kidneys were harvested for morphologic, P-Smad2, renin, neuronal nitric oxide synthase (nNOS) and cyclooxygenase-2 (COX-2) analyses by western and immunohistochemistry at week 0 and 12. Result At week 12, both WT and β6-/- had significantly and similarly increased proteinuria when compared with baseline (P<0.05). β6-/- showed lower systolic blood pressure compared with WT. Although the cortical interstitial volume fraction was similar, β6-/- group had more severe glomerular injury than WT (P<0.05). Ratios of SI vs interstitial fibrosis thus showed disproportionally milder interstitial lesions vs glomerular lesions in β6-/- vs WT. At baseline, β6 -/- did not show altered expression by western blot of P-Smad2, nNOS, renin or COX-2. At week 12 after Nx, β6 -/- micehad decreased P-Smad2 by 5.7-fold and nNOS by 2.4-fold, while renin expression increased 1.8 fold vs WT Nx (P<0.05). There was no difference in COX-2 expression. By immunohistochemistry, nNOS and renin were observed mainly in JGA at week 0 with increased renin extent and intensity and decreased nNOS intensity in β6 -/- vs WT at week 12. Increased COX-2 was detected in macula densa/cTALH and medullary interstitial cells after 5/6 nephrectomy in both WT and β6 -/-.Conclusion We conclude that absence of integrin β6 worsens glomerulosclerosis after 5/6 nephrectomy, with disproportionally milder tubulointerstitial fibrosis at least in part by increasing renin and decreasing nNOS expression in the JGA. We speculate that dysregulated tubuloglomerular feedback due to absent JGA β6 expression in the remnant kidney might be involved in this augmented sclerosis.