Impaired tubular uptake explains albuminuria in early diabetic nephropathy

Abstract

Understanding the pathogenesis of albuminuria in diabetic nephropathy is important to improve methods for early diagnosis and treatment. In this study, we addressed whether albuminuria in diabetes results from altered glomerular filtration and/or altered processing of filtered albumin by the proximal tubule. Type 1 diabetic Munich Wistar rats developed albuminuria after 12 wk of diabetes. Intravital two-photon microscopy revealed similar glomerular permeability in the diabetic and control animals, assessed using both albumin-Alexa568 and 69-kD FITC-dextran; however, diabetic animals demonstrated significantly less filtered fluorescent albumin in renal proximal tubule (PT) cells compared with control animals. We also observed increased albumin-derived urinary peptide excretion in diabetic animals, and hyperglycemia modulated this peptideuria. In conclusion, in the early stages of diabetic nephropathy, the PT plays a major role in the development of albuminuria, which may be preceded by peptideuria.

Figure 1.

Intact urinary albumin excretion is increased in 12-wk diabetic rats. Urinary intact albumin excretion was measured by ELISA for rat serum albumin in 24-h urine samples of 12-wk diabetic rats and age-matched controls. Diabetic rats had significantly increased urinary intact albumin excretion (P < 0.05 control versus diabetic; control n = 4, diabetic n = 4).

Figure 2.

The GSC of albumin and 69-kD FITC-dextran is unchanged in 12-wk diabetic versus control rats. (A) GSC of albumin-Alexa568 in 12-wk diabetic and aged-matched control rats demonstrates no significant change in the GSC for albumin (control n = 4, diabetic n = 4; 13 data points for control animals and 13 data points for diabetic animals over 24 min). (B) GSC of 69-kD FITC-dextran in 12-wk diabetic and aged-matched control rats demonstrates no significant change in the GSC for dextran, confirming the results obtained for albumin GSC (control n = 4, diabetic n = 5; 16 data points for control animals and 12 data points for diabetic animals taken over 24 min). (C) The plasma clearance of albumin-Alexa and 69-kD FITC-dextran over 24 h demonstrates that dextran is quickly cleared from the plasma over 24 h but a large portion of albumin is retained in the plasma despite the two molecules’ having similar GSC (albumin n = 4, dextran n = 4). (D) Representative pictures showing before and 24 min after injection of green 69-kD FITC-dextran (top) and red albumin-Alexa568 (bottom) in 12-wk control rats. Albumin (red) can be seen distributed in PT cells, whereas dextran (green) uptake is negligible. Bar = 30 μm. G, glomerulus; PT, proximal tubule.

Figure 3.

Albumin distribution in PT cells is reduced in diabetes. (A) Representative pictures of the distribution of albumin in the kidney of 12-wk diabetic rats and age-matched control rats. Images shown are before albumin-Alexa568 (red) injection and 2.5 min and 24 min after albumin-Alexa injection. Albumin uptake (red) can be seen in the S1 segment of the PT (arrows). This uptake is dramatically reduced in the diabetic group. Bar = 30 μm. (B) Enlarged picture of the region highlighted by arrows in A. The enlarged picture demonstrates the dramatic change in the distribution of albumin in PT cells. Bar = 15 μm. (C) Quantification of the amount of albumin distributed along the S1 segment throughout the cell (P < 0.01 control versus diabetic; control n = 4, diabetic n = 4). G, glomerulus; PT, proximal tubule.

Figure 4.

Total urinary protein excretion. (A) Total protein excretion in 12-wk diabetic and age-matched control rats demonstrated significantly increased total urinary protein excretion in the diabetic versus control groups (P < 0.01 control versus diabetic; control n = 4, diabetic n = 4). (B) Fractional clearance of [¹⁴C]albumin in 12-wk diabetic rats demonstrates an approximately 2.2-fold increase in total albumin excretion (P < 0.001 control versus diabetic; control n = 7, diabetic n = 9). (C) Total protein excretion (▪) at 4 d after STZ-induced diabetes induction resulted in significantly increased total protein excretion that could be prevented through strict glycemic control (P < 0.05 control versus diabetic; P < 0.05 diabetic versus diabetic + insulin). Analysis of intact albumin excretion by ELISA (□) reveals no significant increase in intact albumin excretion 4 d after STZ-induced diabetes induction despite significant and relatively considerable increases in total protein excretion (control n = 3, diabetic n = 3, diabetic + insulin n = 3). NS, not significant.