Rituximab induces nephrotic syndrome (NS) remission in two-thirds of patients with primary membranous nephropathy (MN), even after other treatments have failed. To assess the relationships among treatment effect, circulating nephritogenic anti-phospholipase A 2 receptor (anti-PLA 2 R) autoantibodies and genetic polymorphisms predisposing to antibody production we serially monitored 24-hour proteinuria and antibody titer in patients with primary MN and long-lasting NS consenting to rituximab (375 mg/m 2 ) therapy and genetic analyses. Over a median (range) follow-up of 30.8 (6.0-145.4) months, 84 of 132 rituximabtreated patients achieved complete or partial NS remission (primary end point), and 25 relapsed after remission. Outcomes of patients with or without detectable anti-PLA 2 R antibodies at baseline were similar. Among the 81 patients with antibodies, lower anti-PLA 2 R antibody titer at baseline (P=0.001) and full antibody depletion 6 months post-rituximab (hazard ratio [HR], 7.90; 95% confidence interval [95% CI], 2.54 to 24.60; P,0.001) strongly predicted remission. All 25 complete remissions were preceded by complete anti-PLA 2 R antibody depletion. On average, 50% anti-PLA 2 R titer reduction preceded equivalent proteinuria reduction by 10 months. Re-emergence of circulating antibodies predicted disease relapse (HR, 6.54; 95% CI, 1.57 to 27.40; P=0.01), whereas initial complete remission protected from the event (HR, 6.63; 95% CI, 2.37 to 18.53; P,0.001). Eighteen patients achieved persistent antibody depletion and complete remission and never relapsed. Outcome was independent of PLA2R1 and HLA-DQA1 polymorphisms and of previous immunosuppressive treatment. Therefore, assessing circulating anti-PLA 2 R autoantibodies and proteinuria may help in monitoring disease activity and guiding personalized rituximab therapy in nephrotic patients with primary MN.

In the present study, we evaluated the effect of simultaneously blocking angiotensin II synthesis and endothelin (ET)-1 activity as a multimodal intervention to implement renoprotection in overt diabetic nephropathy. Mechanisms underlying combined therapy effectiveness were addressed by investigating podocyte structure and function and glomerular barrier size-selective properties. Uninephrectomized rats made diabetic by streptozotocin received orally placebo, lisinopril (12.5 mg/l), the ET(A) receptor antagonist avosentan (30 mg/kg), or their combination from 4 (when animals had proteinuria) to 8 mo. Proteinuria, renal damage, podocyte number, nephrin expression, and glomerular size selectivity by graded-size Ficoll molecule fractional clearance were assessed. Combined therapy normalized proteinuria, provided complete protection from tubulointerstitial damage, and induced regression of glomerular lesions, while only a partial renoprotection was achieved by each drug alone. Lisinopril plus avosentan restored to normal values the number of podocytes. Single therapies only limited podocyte depletion. Defective nephrin expression of diabetes was prevented by each drug. Altered glomerular size selectivity to large macromolecules of diabetic rats was remarkably improved by lisinopril and the combined treatment. Avosentan ameliorated peritubular capillary architecture and reduced interstitial inflammation and fibrosis. The ACE inhibitor and ET(A) receptor antagonist induced regression of glomerular lesions in overt diabetic nephropathy. Regression of renal disease was conceivably the result of the synergistic effect of the ACE inhibitor of preserving glomerular permselective properties and the ET(A) antagonist in improving tubulointerstitial changes. These findings provide mechanistic insights to explain the antiproteinuric effect of this combined therapy in diabetes.

Abstract. Renin-angiotensin system (RAS) inhibitors are effective in reducing renal disease progression in early diabetic nephropathy, but they provide imperfect protection at a later stage. Due to the pivotal role of transforming growth factor-␤ (TGF-␤) in the pathogenesis of diabetic kidney disease, this study tested the effect of simultaneously interrupting TGF-␤ and angiotensin II on disease progression in diabetic rats with overt nephropathy. Diabetes was induced by streptozotocin injection in uninephrectomized rats. Diabetic rats received murine (1D11) or human anti-TGF-␤ monoclonal antibodies alone or in combination with lisinopril, 13C4 irrelevant murine antibody, saline or lisinopril from month 4 (when animals had proteinuria) to month 8. Normal animals served as controls. Systolic BP increase was controlled by single treatments and even more by the combined therapies. 1D11 and lisinopril kept proteinuria at levels numerically lower than irrelevant antibody and saline, while CAT-192 was ineffective. The addition of either TGF-␤ antibody to lisinopril normalized proteinuria. Consistent results were obtained for glomerulosclerosis and tubular damage, which were abrogated by the combined therapy. Interstitial volume expansion and infiltration of lymphocytes/macrophages were limited by 1D11 and lisinopril and further reduced by their combination. The increase of type III collagen in the renal interstitium was partially attenuated by 1D11 and lisinopril while normalized by their combination. It is concluded that anti-TGF-␤ antibody when added to a background of chronic angiotensin-converting enzyme (ACE) inhibition fully arrests proteinuria and renal injury of overt diabetic nephropathy, providing a novel route to therapy and remission of disease for diabetic patients who do not respond to RAS inhibition. Diabetic nephropathy, a major long-term complication of diabetes mellitus, is the most common cause of end-stage renal disease requiring dialysis worldwide (1,2) and is becoming a staggering challenge to public healthcare systems due to the prohibitive costs of renal replacement therapy that could become unaffordable even for developed countries. Typical histologic features characterize diabetic nephropathy, including expansion of the extracellular matrix in the glomerular mesangium, thickening of glomerular and tubular membranes, and tubulointerstitial fibrosis, all of them contributing to the inexorable progressive deterioration of renal function (3).Among treatment options for diabetes, agents that inhibit the renin-angiotensin system (RAS) are particularly effective in reducing renal disease progression (4). This is not simply a function of the effect on systemic and glomerular hypertension, but it can be attributed to the unique property of this class of drugs of limiting excess protein ultrafiltration and its deleterious consequences (5). Angiotensin-converting enzyme inhibitor (ACEi) effectiveness, however, depends on timing of treatment. Experimental data in rats with streptozotocin-induced diabete...

Down-regulation of nephrin and loss of the electron dense structure of slit diaphragm indicate a novel mechanism accounting for proteinuria in diabetic nephropathy. To the extent that glomerular protein trafficking contributes to renal disease progression, our findings may have clinical relevance. Reduction of nephrin in the context of normal expression of CD2AP and podocin can be taken reasonably as a specific marker of renal disease in diabetes. Therapies targeted at correcting podocyte nephrin might be of value for diabetic medicine.

Repertaxin treatment of the recipient animal was effective in preventing granulocyte infiltration and renal function impairment both in syngeneic and in allogeneic settings. The possibility to modulate I/R injury in this rat model opens new perspectives for preventing posttransplant delayed graft function in humans.

Today angiotensin II inhibition is primarily used to slow the rate of progression of kidney diseases. There is evidence that these therapies can induce a partial regression of glomerular lesions. However, we do not know yet the extent of sclerotic lesion regression and whether new glomerular tissue is formed to help support the renal function. We used male Munich Wistar Fromter (MWF) rats, an experimental model for progressive kidney disease, to quantify kidney structural lesions upon angiotensin-converting enzyme (ACE) inhibition therapy. Animals were studied at 50 weeks of age, when renal function and structure are severely altered, and after a 10-week observation period, without or with treatment with lisinopril (80 mg/l in drinking water). A group of untreated Wistar rats was used as controls. With age, proteinuria, and serum creatinine worsen, but lisinopril almost normalized proteinuria and stabilized serum creatinine. Serial section analysis of whole glomerular tufts showed that at baseline, glomerulosclerosis affected the entire glomerular population, and that these changes further increased with age. Lisinopril significantly reduced incidence and extent of glomerulosclerosis, with the presence of glomerular tufts not affected by sclerosis (23% of glomeruli). Glomerular volume was not significantly affected by treatment, and glomerular mass spared from sclerosis increased from 46.9 to 65.5% upon treatment, indicating consistent regeneration of glomerular tissue. Lisinopril normalized baseline glomerular transforming growth factor-beta and alpha-smooth muscle actin overexpression, and prevented worsening of interstitial changes. Hence, ACE inhibition, which is widely used in human kidney disease, may not only halt the progression of renal failure, but also actually induce the regeneration of new renal tissue.

We previously reported that angiotensin-converting enzyme inhibitor (ACEi) renoprotection in Munich Wistar Frömter (MWF) rats, which develop progressive glomerular injury, was associated with podocyte repopulation and preservation of glomerular architecture. Here, we studied the time course of the lesions, their cellular components, and the effect of ACEi. Early glomerular lesions were synechiae, followed by extracapillary crescents and glomerulosclerosis. The majority of cells forming crescents were claudin1 ؉ parietal epithelial cells and, to a lesser extent, WT1؉ podocytes, both in active proliferation. In crescents, cells expressing the metanephric mesenchyme marker NCAM were also found. Three distinct populations of parietal epithelial cells were identified in the rat Bowman's capsule: NCAM ؉ WT1 ؊ cells, also expressing progenitor cell marker CD24, and NCAM ؉ WT1 ؉ and NCAM ؊ WT1 ؉ cells, the latter population representing parietal podocytes. After exposure to inductive medium, cultured parietal epithelial cells that were obtained by capsulated glomeruli generated podocytes, documenting their progenitor nature. Mitotic activity of cultured renal progenitors was induced by angiotensin II through the down-regulation of cell cycle inhibitor C/EBP␦ expression. Treatment with ACEi reduced number and extension of crescents and glomerulosclerosis in MWF rats. Renoprotection was accomplished through the limitation of NCAM ؉ progenitor proliferation via the modulation of C/EBP␦. Thus, chaotic migration and proliferation of the Bowman's capsule progenitor cells pave the way to crescent formation and subsequent sclerosis. ACEi, by moderating progenitor cell activation, restores glomerular architecture and prevents renal disease progression.

Angiotensin-converting enzyme (ACE) inhibition induces glomerular repair in the Munich WistarFrömter (MWF) rat, a model of spontaneous glomerular injury. In this study, we investigated whether this effect is related to changes in glomerular cell number, particularly of podocytes, which are progressively lost with age. MWF rats with advanced nephropathy were studied at both 40 weeks and after 20 weeks of observation either with or without treatment with the ACE inhibitor lisinopril. Forty-weekold Wistar rats were used as controls. In untreated MWF rats, proteinuria, hypertension, glomerulosclerosis, and renal function worsened, while lisinopril induced regression of both functional and structural changes. Despite glomerular hypercellularity in untreated MWF rats, the number of endothelial cells per glomerulus did not change, and podocyte number even decreased. ACE inhibition halted the progressive increase in glomerular cell number and enhanced endothelial cell volume density. Surprisingly, lisinopril not only halted age-related podocyte loss but also increased the number of glomerular podocytes above baseline, which was associated with an increased number of proliferating Wilms tumor 1-positive cells, loss of cyclin-dependent kinase inhibitor p27 expression, and increased number of parietal podocytes. These data indicate that ACE inhibition restructures glomerular capillary , primarily by restoring the podocyte population in this model of glomerular injury. Increased parietal podocyte number in lisinopril-treated MWF rats suggests that the remodeling of Bowman's capsule epithelial cells contributes to this effect.