Podocyte vascular endothelial growth factor (Vegf164) overexpression causes severe nodular glomerulosclerosis in a mouse model of type 1 diabetes.

AIMS/HYPOTHESIS: The pathogenic role of excessive vascular endothelial growth factor (VEGF)-A in diabetic nephropathy has not been defined. We sought to test whether increased podocyte VEGF-A signalling determines the severity of diabetic glomerulopathy. METHODS: Podocyte-specific, doxycycline-inducible Vegf164 (the most abundant Vegfa isoform) overexpressing adult transgenic mice were made diabetic with low doses of streptozotocin and examined 12 weeks after onset of diabetes. We studied diabetic and non-diabetic transgenic mice fed a standard or doxycycline-containing diet. VEGF-A and albuminuria were measured by ELISA, creatinine was measured by HPLC, renal morphology was examined by light and electron microscopy, and gene expression was assessed by quantitative PCR, immunoblotting and immunohistochemistry. RESULTS: Podocyte Vegf164 overexpression in our mouse model of diabetes resulted in advanced diabetic glomerulopathy, characterised by Kimmelstiel-Wilson-like nodular glomerulosclerosis, microaneurysms, mesangiolysis, glomerular basement membrane thickening, podocyte effacement and massive proteinuria associated with hyperfiltration. It also led to increased VEGF receptor 2 and semaphorin3a levels, as well as nephrin and matrix metalloproteinase-2 downregulation, whereas circulating VEGF-A levels were similar to those in control diabetic mice. CONCLUSIONS/INTERPRETATION: Collectively, these data demonstrate that increased podocyte Vegf164 signalling dramatically worsens diabetic nephropathy in a streptozotocin-induced mouse model of diabetes, resulting in nodular glomerulosclerosis and massive proteinuria. This suggests that local rather than systemic VEGF-A levels determine the severity of diabetic nephropathy and that semaphorin3a signalling and matrix metalloproteinase-2 dysregulation are mechanistically involved in severe diabetic glomerulopathy.

Glomerular injury is exacerbated in diabetic integrin alpha1-null mice.

Excessive glomerular collagen IV and reactive oxygen species (ROS) production are key factors in the development of diabetic nephropathy. Integrin alpha1beta1, the major collagen IV receptor, dowregulates collagen IV and ROS production, suggesting this integrin might determine the severity of diabetic nephropathy. To test this possibility, wild-type and integrin alpha1-null mice were rendered diabetic with streptozotocin (STZ) (100 mg/kg single intraperitoneal injection), after which glomerular filtration rate (GFR), glomerular collagen deposition, and glomerular basement membrane (GBM) thickening were evaluated. In addition, ROS and collagen IV production by mesangial cells as well as their proliferation was measured in vitro. Diabetic alpha1-null mice developed worse renal disease than diabetic wild-type mice. A significant increase in GFR was evident in the alpha1-null mice at 6 weeks after the STZ injection; it started to decrease by week 24 and reached levels of non-diabetic mice by week 36. In contrast, GFR only increased in wild-type mice at week 12 and its elevation persisted throughout the study. Diabetic mutant mice also showed increased glomerular deposition of collagen IV and GBM thickening compared to diabetic wild-type mice. Primary alpha1-null mesangial cells exposed to high glucose produced more ROS than wild-type cells, which led to decreased proliferation and increased collagen IV synthesis, thus mimicking the in vivo finding. In conclusion, this study suggests that lack of integrin alpha1beta1 exacerbates the glomerular injury in a mouse model of diabetes by modulating GFR, ROS production, cell proliferation, and collagen deposition.

Reabsorption of betaine in Henle's loops of rat kidney in vivo.

This study was designed 1) to localize and 2) to characterize betaine reabsorption from the tubular lumen in rat kidney in vivo, and 3) to test whether reabsorption is modulated by the diuretic state. [(14)C]betaine (+ [(3)H]inulin) was microperfused through the proximal convoluted tubule (PCT) and microinfused into late proximal (LP) and early distal (ED) tubules, long loops of Henle (LLH), and vasa recta of the rat in vivo et situ, and the fractional recovery of the (14)C label was determined end proximally (PCT) and in the final urine, respectively. [(14)C]betaine was not reabsorbed during ED microinfusion, whereas fractional reabsorption during LP microinfusion was 82% at 0.06 mM betaine and decreased gradually to 4.8% at 60 mM. L-Proline had lower Michaelis-Menten constant (K(m)) and sarcosine a higher K(m) than betaine. Chronic, but not acute, diuresis inhibited betaine reabsorption in Henle's loops. Fractional [(14)C]betaine reabsorption in PCT was much smaller than that during LP microinfusion. [(14)C]betaine (7.28 mM) microinfused 1) into LLH was reabsorbed to 30% and 2) into vasa recta appeared in the ipsilateral urine to a much higher extent than contralaterally. In both cases, no saturation was detected at 70 mM. We conclude that betaine is reabsorbed by mediated transport from descending limbs of short Henle's loops by a proline-preferring carrier in a diuresis-modulated manner. In the deep medulla, bidirectional blood/urine betaine transport exists.

Kidney-targeted liposome-mediated gene transfer in mice.

To develop gene therapy targeted to the kidney, we compared three different routes of liposome-mediated gene delivery to the kidney in mice, ie intra-renal-pelvic, intra-renal-arterial, and intra-renal-parenchymal injections. A plasmid construct, pCMV beta gal, containing a cytomegalovirus (CMV) immediate-early gene promoter and a beta-galactosidase reporter gene was mixed with a 1:1 liposome mixture of N[1-(2,3-dioleoyloxy)propyl]-N,N,trimethylammonium chloride (DOTMA)/dioleoyl phosphatidyl ethanolamine (DOPE). The pCMV beta gal-liposome complex was injected into the left kidney via three different routes. The efficacy of gene transfer was assessed using 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) staining on frozen kidney sections 3 to 42 days after injections. Cells with beta-galactosidase activity were detected in the cortex and outer medulla in both intra-renal-pelvic and intra-renal-arterial groups, but not in the intra-renal-parenchymal group or in the contralateral noninjected kidney. Evidence of gene transfer was observed only in tubular epithelial cells, but not in glomerular, vascular, or interstitial compartments. The levels of beta-galactosidase expression started to decrease 3 weeks after injection. The gene transfer in the kidney was not associated with nephrotoxicity as assessed by blood urea nitrogen levels and renal histology. We conclude that both intra-renal-pelvic and intra-renal-arterial injections provide a transient gene transfer to the renal tubular cells and are suitable routes for kidney-targeted gene therapy.

Distribution of de novo synthesized betaine in rat kidney: role of renal synthesis on medullary betaine accumulation.

The trimethylamine glycine-betaine is accumulated to high concentrations in medullary cells of mammalian kidneys, whereas betaine synthesis from choline is predominant in the renal cortex. We investigated the contribution of renal betaine synthesis to medullary betaine accumulation. De novo synthesis of betaine in situ was accomplished by injecting [14C]choline into the renal artery of male Sprague-Dawley rats. [14C]betaine was measured in the renal cortex and medulla, as well as in serum and urine samples. Betaine concentration in the cortex decreased from 3.5 +/- 1.3 at 5 min to 0.4 +/- 0.2 nmol/mg protein at 60 min, but it increased from 1.4 +/- 0.1 to 2.5 +/- 0.6 nmol/mg protein in the medulla. Serum and total urine [14C]betaine increased from 2.7 +/- 1.3 and 0.9 +/- 0.1 nmol/ml at 5 min to 5.3 +/- 0.3 and 2.1 +/- 0.4 nmol/ml at 60 min, respectively. Concentrations of newly synthesized betaine were not decreased by the ligation of the hepatic artery and portal vein, suggesting that most [14C]betaine was synthesized in the kidney. Coinjection with 5 mM dimethylamino-ethanol, a choline oxidase inhibitor, and 100 mM cold betaine reduced medullary betaine accumulation by 80 and 76%, respectively. Water deprivation for 60 h increased both cortical and medullary [14C]betaine, whereas furosemide diuresis decreased the medullary [14C]betaine concentration. We concluded that betaine synthesized in the kidney can be accumulated in the medulla and that the medullary concentrations of newly synthesized betaine are closely related to the hydration state of the animal.

Kinetics and osmoregulation of Na(+)-and Cl(-)-dependent betaine transporter in rat renal medulla.

Betaine is one of the major organic osmolytes that accumulate in the renal medulla in response to high extracellular tonicity. Recent studies in MDCK cells have shown that betaine is taken up by an Na(+)- and Cl(-)-dependent transporter located on the basolateral membrane. We demonstrate here the presence of Na(+)-Cl(-)-dependent betaine transporter(s) in tubule suspensions prepared from the rat outer and inner medulla. The betaine transport activity was two to three times higher in the inner medulla compared with the outer medulla. The removal of Na+ and Cl- reduced betaine uptake in the outer medullary tubules by 86% and 82%, respectively. The betaine uptake was decreased by 39% in hypotonic buffer (189 mosmol/ kgH2O) and increased by 82% in hypertonic buffer (545 mosmol/kgH2O), compared with isotonic buffer (308 mosmol/ kgH2O). Kinetic studies of Na(+)-dependent betaine uptake in the outer medullary tubules revealed both a low- and a high-affinity component as follows: low-affinity and high volume component with Michaelis constant (K(m)1) of 8.6 mM and maximal uptake rate (Vmax1) of 112 pmol.microgram protein-1.h-1; and a low-volume and high-affinity component with K(m)2 of 0.141 mM and Vmax2 of 10 pmol. microgram protein-1.h-1. To investigate whether the Na(+)-Cl(-)-dependent betaine transporter is regulated by tonicity in vivo, we quantitated its mRNA in rat renal cortex and outer and inner medulla using both canine and rat Na(+)-Cl(-)-dependent betaine transporter cDNA probes. A single band of 3.0 kb was seen in the Northern blots prepared from both outer and inner medulla, but not in the cortex. Water deprivation for 3 days increased the abundance of this mRNA in the outer and inner medulla by 140% and 170%, respectively, but did not affect its expression in the cortex. In conclusion, Na(+)-Cl(-)-dependent betaine transporter(s) is present in rat outer and inner medullary tubules, and betaine transporter mRNA abundance is regulated by the hydration state in vivo.

Bicarbonate dependency of betaine synthesis in cultured LLC-PK1 cells.

Betaine, one of the major renal organic osmolytes, is synthesized from choline by choline dehydrogenase (EC 1.1.99.1) and betaine-aldehyde dehydrogenase (EC 1.2.1.8) in the kidney. A recent in vitro study has shown that betaine synthesis by renal cortical homogenate is dependent on millimolar amounts of bicarbonate. The present study was aimed to investigate the bicarbonate dependency of betaine formation in cultured LLC-PK1 cells. The data show that betaine formation increases in accordance with a rise in extracellular bicarbonate levels. The measured quantities of [14C]betaine synthesis ranged from 13.4 +/- 1.5 (4.6 mM HCO3-) to 38.0 +/- 1.4 pmol.micrograms protein-1.h-1 (24 mM HCO3-). The carbonic anhydrase inhibitor acetazolamide, added to the incubation medium to block bicarbonate transport, reduced betaine synthesis from choline by 41-49%. We conclude that betaine synthesis in LLC-PK1 cells is dependent on extracellular bicarbonate levels and is reduced by the inhibition of carbonic anhydrase. Because betaine accumulates in renal medulla during antidiuresis, our observations suggest a possible link between acid-base homeostasis and concentration mechanisms in the kidney.