Mauro Abbate

Mesenchymal Stem Cells Are Renotropic, Helping to Repair the Kidney and Improve Function in Acute Renal Failure

Injury to a target organ can be sensed by bone marrow stem cells that migrate to the site of damage, undergo differentiation, and promote structural and functional repair. This remarkable stem cell capacity prompted an investigation of the potential of mesenchymal and hematopoietic stem cells to cure acute renal failure. The model of renal injury induced in mice by the anticancer agent cisplatin was chosen. Injection of mesenchymal stem cells of male bone marrow origin remarkably protected cisplatin-treated syngeneic female mice from renal function impairment and severe tubular injury. Y chromosome-containing cells localized in the context of the tubular epithelial lining and displayed binding sites for Lens culinaris lectin, indicating that mesenchymal stem cells engraft the damaged kidney and differentiate into tubular epithelial cells, thereby restoring renal structure and function. Mesenchymal stem cells markedly accelerated tubular proliferation in response to cisplatin-induced damage, as revealed by higher numbers of Ki-67-positive cells within the tubuli with respect to cisplatin-treated mice that were given saline. Hematopoietic stem cells failed to exert beneficial effects. These results offer a strong case for exploring the possibility that mesenchymal stem cells by virtue of their renotropic property and tubular regenerative potential may have a role in the treatment of acute renal failure in humans.

Insulin-Like Growth Factor-1 Sustains Stem Cell–Mediated Renal Repair

In mice with cisplatin-induced acute kidney injury, administration of bone marrow-derived mesenchymal stem cells (MSC) restores renal tubular structure and improves renal function, but the underlying mechanism is unclear. Here, we examined the process of kidney cell repair in co-culture experiments with MSC and cisplatin-injured proximal tubular epithelial cells (PTEC). Exposure of PTEC to cisplatin markedly reduced cell viability at 4 days, but co-culture with MSC provided a protective effect by promoting tubular cell proliferation. This effect was mediated by insulin-like growth factor-1 (IGF-1), highly expressed by MSC as mRNA and protein, since blocking the growth factors function with a specific antibody attenuated cell proliferation of PTEC. Confirming this, knocking down IGF-1 expression in MSC by small interfering-RNA also resulted in a significant decrease in PTEC proliferation and increased apoptosis. Furthermore, in the murine model of cisplatin-induced kidney injury, administering IGF-1 gene-silenced MSC limited their protective effect on renal function and tubular structure. These findings indicate that MSC exert beneficial effects on tubular cell repair in acute kidney injury by producing the mitogenic and pro-survival factor IGF-1.

Rituximab in Idiopathic Membranous Nephropathy: A One-Year Prospective Study

Currently available monoclonal antibodies against the B cell surface antigen CD20 have been employed to explore whether specific inhibition of B cells may help improve the outcome of idiopathic membranous nephropathy (IMN) and may avoid the side effects of steroids and immunosuppressants. This prospective, observational study evaluated the 1-yr outcome of eight IMN patients with persistent (>6 mo) urinary protein excretion > 3.5 g/24 h given four weekly infusions of the anti-CD20 antibody rituximab (375 mg/m(2)). At 3 and 12 mo, proteinuria significantly decreased from mean (+/- SD) 8.6 +/- 4.2 to 4.3 +/- 3.3 (-51%, P < 0.005) and 3.0 +/- 2.5 (-66%, P < 0.005) g/24 h, albumin fractional clearance from 2.3 +/- 2.1 to 1.2 +/- 1.7 (-47%, P < 0.05) and 0.5 +/- 0.6 (-76%, P < 0.003), and serum albumin concentration increased from 2.7 +/- 0.5 to 3.1 +/- 0.3 (+21%, P < 0.05) and 3.5 +/- 0.4 (+41%, P < 0.05) mg/dl. At 12 mo, proteinuria decreased to < or =0.5 g/24 h or < or =3.5 g/24 h in two and three patients, respectively. Proteinuria decreased in the remaining patients by 74%, 44%, and 41%, respectively. Body weight, diastolic BP, and serum cholesterol progressively decreased in parallel with an improvement of edema in all patients. Renal function stabilized (Delta1/creatinine: +0.002 +/- 0.007). CD20 B lymphocytes fell below normal ranges up to study-end. No patient had major drug-related events or major changes in other laboratory parameters. Rituximab thus promotes sustained disease remission in patients otherwise predicted to progress to ESRD, and it is safe. The long-term risk/benefit profile of this novel, disease-specific approach seems much more favorable to that of commonly employed immunosuppressive drugs.

Add-On Anti–TGF-β Antibody to ACE Inhibitor Arrests Progressive Diabetic Nephropathy in the Rat

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-beta (TGF-beta) in the pathogenesis of diabetic kidney disease, this study tested the effect of simultaneously interrupting TGF-beta 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 (CAT-192) anti-TGF-beta 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-beta 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-beta 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.

How To Fully Protect the Kidney in a Severe Model of Progressive Nephropathy: A Multidrug Approach

The current therapy for chronic proteinuric nephropathies is angiotensin-converting enzyme inhibitors (ACEi), which slow, but may not halt, the progression of disease, and which may be not effective to the same degree in all patients. In accelerated passive Heymann nephritis (PHN), this study assessed the effect of combining ACEi with angiotensin II receptor antagonist (AIIRA) and with statin that, besides lowering cholesterol, influences inflammatory and fibrogenic processes. Uninephrectomized PHN rats were divided into four groups (n = 10 each) and daily given oral doses of the following: vehicle; 40 mg/L lisinopril; 100 mg/L lisinopril plus L-158,809; 0.3 mg/kg lisinopril plus L-158,809 plus cerivastatin. Treatments started at 2 mo when rats had massive proteinuria and signs of renal injury and lasted until 10 mo. Increases in BP were equally lowered by treatments. ACEi kept proteinuria at levels comparable to pretreatment and numerically lower than vehicle. The addition of AIIRA to lisinopril was more effective, being proteinuria reduced below pretreatment values and significantly lower than vehicle. When cerivastatin was added on top of ACE inhibition and AIIR blockade, urinary protein regressed to normal values and renal failure was prevented. Renal ACE activity was increased threefold in PHN, it was inhibited by more than 60% after ACEi, and decreased below control values with triple therapy. Cerivastatin inhibited ACE activity by 30%. Glomerulosclerosis, tubular damage and interstitial inflammation were ameliorated by ACEi alone or combined with AIIRA, and prevented by addition of statin. TGF-beta(1) mRNA upregulation in PHN kidney was partially reduced after ACEi or combined with AIIRA and almost normalized after adding statin. Cerivastatin inhibited TGF-beta(1) gene upregulation by 25%. These data suggest a possible future strategy to induce remission of proteinuria, lessen renal injury, and protect from loss of function in those patients who do not fully respond to ACEi therapy.

Protein Overload Induces Fractalkine Upregulation in Proximal Tubular Cells through Nuclear Factor κB– and p38 Mitogen-Activated Protein Kinase–Dependent Pathways

Investigated was the effect of high albumin concentrations on proximal tubular cell expression of fractalkine. Human proximal tubular cells (HK-2) were incubated with human serum albumin (HSA), which induced a dose-dependent increase in fractalkine mRNA associated with increased levels of both membrane-bound and soluble forms of the protein. To evaluate the role of nuclear factor kappaB (NF-kappaB) activation in HSA-induced fractalkine mRNA, HK-2 cells were infected with a recombinant adenovirus encoding the natural inhibitor of NF-kappaB, IkBalpha; a 43% reduction of fractalkine mRNA levels resulted. Similarly, when cells were infected with the recombinant adenovirus expressing dominant negative mutant of the IkB kinase 2, a 55% inhibition of fractalkine mRNA was achieved. p38 mitogen-activated protein kinase was activated by HSA and was involved in NF-kappaB-dependent transcription of fractalkine. In kidneys of mice with bovine serum albumin overload proteinuria, fractalkine mRNA levels were 2.3-fold greater than those of controls. Fractalkine expression was also induced in tubular epithelial cells in this model. Anti-CXCR1 antibody treatment limited interstitial accumulation of mononuclear cells. Protein overload is a promoter of fractalkine gene induction mediated by NF-kappaB and p38 activation in proximal tubular cells. Fractalkine might contribute to direct mononuclear cells into peritubular interstitium and enhance their adhesion property, which in turn would favor inflammation and disease progression.

Sirolimus versus cyclosporine therapy increases circulating regulatory T cells, but does not protect renal transplant patients given alemtuzumab induction from chronic allograft injury.

Background. In kidney transplant recipients with alemtuzumab induction maintained on mycophenolate mofetil (MMF) immunosuppression, sirolimus (SRL) promotes significant expansion of circulating CD4+CD25high regulatory T cells (Treg). This might translate into more effective protection against chronic graft injury compared to cyclosporin A (CsA), which, in the same clinical setting, does not affect Treg. Methods. To assess this hypothesis, in the extension of a single-center, prospective, randomized, open, blind endpoint study aimed to assess the effect of low-dose SRL or CsA on circulating Treg, we compared the outcomes of renal transplant recipients on SRL (n=11) or CsA (n=10) by per-protocol biopsies and serial measurements of glomerular filtration rate (GFR), renal plasma flow (RPF), and 24-hour proteinuria over 30 months posttransplant. Results. Despite 4-fold higher CD4+CD25high Treg counts (22.1±12.2% vs. 5.7±4.2% of CD3+CD4+ T cells), SRL-treated patients, compared to CsA-treated patients, had a significantly higher tubular C4d staining score (1.1±0.6 vs. 0.2±0.3, P<0.01), with nonsignificant trends to higher chronic allograft damage index score (5.6±2.4 vs. 3.7±3.3), faster GFR (−2.92±0.33 vs. −0.28±0.44 ml/min/1.73m2 per year), and RPF (−10.80±5.45 vs. −1.86±3.09 ml/min/1.73 m2 per year) decline, and more clinical proteinuria (n=6 vs. 4). There was no significant correlation between Treg counts and any considered outcome variable in the study group as a whole and within each cohort. Conclusions. These data suggest that, despite enhanced Treg expression, low-dose SRL combined to alemtuzumab induction and MMF-based steroid-free maintenance therapy, does not appreciably protect renal transplant recipients from chronic allograft injury and dysfunction.

Rituximab for Idiopathic Membranous Nephropathy: Who Can Benefit?

Rituximab effectively reduces proteinuria in patients with idiopathic membranous nephropathy (IMN), but response to treatment may vary from patient to patient. The association between baseline clinical, laboratory, and histology covariates and proteinuria reduction was evaluated retrospectively by multiple linear regression analysis at 3 mo after rituximab therapy in 14 patients with IMN with proteinuria > 3.5 g/24 h while on angiotensin-converting enzyme inhibition for at least 6 mo and no previous remissions. The association strength was expressed by standardized beta coefficients (SbetaC). Glomerular (SbetaC = 0.48, P = 0.049) and tubulointerstitial (TI) scores (SbetaC = 0.61, P = 0.003) predicted the outcome. Among glomerular and TI score components, tubular atrophy (SbetaC = 0.59, P = 0.003) and interstitial fibrosis (SbetaC = 0.60, P = 0.001) were significantly associated with 3-mo proteinuria. Urinary protein excretion decreased from 9.1 +/- 4.0 to 4.6 +/- 3.5 g/24 h (P < 0.001) in eight patients with TI score 1.7 but did not change in six with a score > or = 1.7. Nine additional patients with IMN then were allocated prospectively to rituximab treatment on the basis of a TI score < 1.7. Three-month proteinuria decreased in all patients from 8.9 +/- 5.3 to 4.9 +/- 3.9 g/24 h (P < 0.001) and serum albumin increased from 2.2 +/- 0.6 to 2.8 +/- 0.5 mg/dl (P < 0.01). Changes in serum albumin and cholesterol were inversely correlated (P < 0.02, r = -0.44). Rituximab achieved CD20 and CD19 depletion in all patients. In patients with IMN and nephrotic proteinuria despite angiotensin-converting enzyme inhibition therapy, renal biopsy findings may help in predicting response to rituximab and defining selection criteria for randomized trials that aim to assess the risk/benefit profile of B cell target therapy as compared with aspecific immunosuppressants and/or conservative therapy alone.

Expression of NCAM recapitulates tubulogenic development in kidneys recovering from acute ischemia

Recovery of the kidney from acute renal failure relies on a sequence of events including epithelial cell dedifferentiation and proliferation followed by differentiation and restoration of the functional integrity of the nephron. The factors responsible for, and the significance of, reversion to a less differentiated cell phenotype and its relationship to the proliferative response after ischemia are poorly understood. In an attempt to identify adhesion molecules that may be influential in the recovery process, the expression of neural cell adhesion molecule (NCAM) and markers of epithelial differentiation and proliferation were analyzed at various times after an ischemic insult. In maturing nephrons, NCAM is detectable by immunohistochemistry in renal vesicles, S-shaped bodies, and early tubules. There is minimal cellular NCAM expression in normal tubules of the adult kidney. In contrast, in postischemic kidneys, NCAM expression is abundant in S3 proximal tubule cells 5 days after reperfusion. As in developing tubules, NCAM is concentrated in basal and lateral aspects of cells that have no apical gp330 or dipeptidyl peptidase IV detectable on their brush border. The expression of NCAM is preceded by disassembly of the brush border and proliferation of surviving S3 cells, which is most prominent at 2 days postischemia. NCAM expression persists in some flattened and dedifferentiated cells for up to 7 wk after ischemia. Thus proximal tubule epithelial cells of the postischemic kidney express NCAM in a pattern that recapitulates the expression of NCAM in the developing kidney. Such reversion of phenotype extends at least back to the early stages of renal vesicle formation, and this reversion may represent a critical step in the reestablishment of a normal tubule. NCAM-matrix interactions may mediate the motogenic and mitogenic responses of the dedifferentiated epithelium that are critical to reestablishment of a functional proximal tubule.Recovery of the kidney from acute renal failure relies on a sequence of events including epithelial cell dedifferentiation and proliferation followed by differentiation and restoration of the functional integrity of the nephron. The factors responsible for, and the significance of, reversion to a less differentiated cell phenotype and its relationship to the proliferative response after ischemia are poorly understood. In an attempt to identify adhesion molecules that may be influential in the recovery process, the expression of neural cell adhesion molecule (NCAM) and markers of epithelial differentiation and proliferation were analyzed at various times after an ischemic insult. In maturing nephrons, NCAM is detectable by immunohistochemistry in renal vesicles, S-shaped bodies, and early tubules. There is minimal cellular NCAM expression in normal tubules of the adult kidney. In contrast, in postischemic kidneys, NCAM expression is abundant in S3 proximal tubule cells 5 days after reperfusion. As in developing tubules, NCAM is concentrated in basal and lateral aspects of cells that have no apical gp330 or dipeptidyl peptidase IV detectable on their brush border. The expression of NCAM is preceded by disassembly of the brush border and proliferation of surviving S3 cells, which is most prominent at 2 days postischemia. NCAM expression persists in some flattened and dedifferentiated cells for up to 7 wk after ischemia. Thus proximal tubule epithelial cells of the postischemic kidney express NCAM in a pattern that recapitulates the expression of NCAM in the developing kidney. Such reversion of phenotype extends at least back to the early stages of renal vesicle formation, and this reversion may represent a critical step in the reestablishment of a normal tubule. NCAM-matrix interactions may mediate the motogenic and mitogenic responses of the dedifferentiated epithelium that are critical to reestablishment of a functional proximal tubule.

Effects of Rituximab on Morphofunctional Abnormalities of Membranous Glomerulopathy

BACKGROUND AND OBJECTIVESnIn idiopathic membranous nephropathy (IMN), CD(20) B-cell depletion by rituximab may induce nephrotic syndrome (NS) remission. Whether this is associated with kidney function restoration and regression of the glomerular pathology was evaluated.nnnDESIGN, SETTING, PARTICIPANTS, & MEASUREMENTSnTreatment-induced morphofunctional changes were evaluated in 7 IMN patients consenting to repeat functional and morphologic evaluations after stable disease remission achieved by four weekly rituximab (375 mg/m(2)) infusions.nnnRESULTSnOver a median of 21 mo from rituximab administration, NS remission was associated with 8.5-fold increase versus baseline in sodium fractional clearance from 1.56 to 13.25, decrease in renal plasma flow from 440.8 to 276.6 ml/min per 1.73 m(2), stable glomerular filtration rate, and increased renal vascular resistances. Changes in sodium fractional clearance and hemoglobin concentration were positively correlated (r = 0.82). Biopsy reevaluations showed complete or partial reabsorption of subepithelial deposits. Median (interquartile range) IgG4 staining score decreased from 3 (3-3) to 1 (0-2), whereas total numbers of slit diaphragms (0.27; range, 0.19 to 0.30 versus 0.86; range, 0.53 to 1.16 slits/mum glomerular basement membrane) and percentages of those with electron-dense diaphragm (55.2; range, 42.0 to 62.0 versus 78.5; range, 73.0 to 82.7 of all slits) significantly increased in parallel with amelioration of glomerular ultrastructural changes. Changes in slit frequency and albumin fractional clearance were negatively correlated (r = -0.79).nnnCONCLUSIONSnIn human IMN, treatment-induced NS remission is associated with restoration of sodium homeostasis and kidney hemodynamics, and regression of the glomerular changes underlying proteinuria. These effects are likely to translate into long-term renoprotection.