Biochemical-molecular markers in unilateral ureteral obstruction

Congenital obstructive nephropathy is the primary cause of end-stage renal disease in children. Rapid diagnosis and initiation of the treatment are vital to preserve function and/or to slow down renal injury. Obstructive uropathy effects -decline in the plasmatic renal flow and glomerular filtration rate, interstitial infiltrate of leukocytes, significant decrease of the urine concentration, loss of the capacity to concentrate urine as well as fibrosis and apoptosis- are a consequence of a variety of factors that work in complex ways and are still not fully understood. Mediators as angiotensin II, transforming growth factor-beta(TGF-beta) and nitric oxide (NO) have been implicated in congenital obstructive nephropathy. The renin-angiotensin system is regulated in different ways, affecting both renal structure and function, and that it in turn depends upon the duration of the obstruction. On the other hand, the role of nitric oxide in renal injury remains somewhat controversial due to the fact that it can exert opposite effects such as cytoprotective and prooxidant / proapoptotic efects as well as proinflammatory and anti-inflammatory effects. In addition, reactive oxidative species (ROS) might contribute to the progression of renal disease. During unilateral ureteral obstruction induced uncoordinated and aberrant growth may lead to the loss of cellular phenotype and apoptosis. Promoting inflammatory responses, the oxidizers can regulate the adherence of certain molecules and proinflammatory mediators, transcription factors and fibrogenic cytokines, that are clearly involved in the progression of renal disease. The congenital obstructive nephropathy is characterized by tubular atrophy, cellular proliferation, apoptosis and fibrosis; immature kidney is more susceptible than adult kidney to showing the above mentioned alterations.

Effect of glandular kallikrein on distal bicarbonate transport. Role of basolateral Cl-/HCO3- exchanger and vacuolar H(+)-ATPase

The luminal membrane of collecting duct cells, specially the intercalated cells, is normally exposed to active kallikrein. This is due to the specific localization of renal kallikrein in the connecting tubule cells. We have previously reported inhibition of distal bicarbonate secretion by renal kallikrein. The present study was performed to evaluate the participation of basolateral Cl-/HCO3- exchanger and luminal H(+)-ATPase activity of cortical collecting duct segments (CCD) in the mechanism involved in the inhibition of bicarbonate secretion induced by the enzyme. The effect of orthograde injections of 1 microgram/ml (250 U/6.3 mg) pig pancreatic kallikrein, in the absence and presence of 1 mM DIDS (stilbene-disulfonic acid) in the renal tubule system, was evaluated. Urine fractions were collected after two-minutes stop-flow. Changes in the urine fraction (Fr) related to those in free-flow urine samples (Ff) were related to the respective polyfructosan (Inutest) ratio. Renal kallikrein activity (Fr:Ff kallikrein/Fr:Ff polyfructosan) increased significantly in the first 120 microliters urine fraction collected after glandular 1 microgram/ml kallikrein, P < 0.05, (first stop-flow) and after glandular 1 microgram/ml kallikrein plus 1 mM. DIDS P < 0.05 (second stop flow). Bicarbonate secretion rate (Fr:Ff HCO3-/Fr:Ff polyfructosan) of collecting ducts was significantly reduced in the first 120 microliters urine fraction collected, related to control, during the first and second stop-flow periods. No difference was shown in bicarbonate excretion between the first 120 microliters urine fractions collected after administration of glandular kallikrein and glandular kallikrein plus DIDS. To measure H(+)-ATPase activity, rat microdissected cortical collector tubules (CCD) were incubated in the presence of increasing glandular kallikrein doses (A: 93, B: 187 and C: 375 mU/200 microL) in the presence of ouabain (4 microM) and omeprazole (100 microM) to inhibit Na(+)-K(+)-ATPase and H(+)-K(+)-ATPase, respectively. In CCD, bafilomycin-sensitive H(+)-ATPase activity (pmol/mm/min) after increasing kallikrein doses did not differ significantly from control...

Efectos de la administración de dosis crecientes de kalikreína sobre la secreción de bicarbonato en nefrón distal / Effect of the administration of increasing doses of kallikrein on bicarbonate secretion on distal nephron

La localización de vesículas secretoras de kalikreína renal en contacto con la membrana apical en el túbulo conector permite asumir la presencia de la enzima activa en la membrana luminal de segmentos distales a su sitio de secreción. Los presentes experimentos fueron realizados a fin de demostrar la hipótesis de que la kalikreína renal está involucrada en el transporte de bicarbonato en el segmento correspondiente a túbulo colector cortical. La administración intraluminal de kalikreína glandular (1 y 3 µ/ml) por arteria renal hacia el sistema tubular de riñón, con interrupción simultánea del flujo sanguíneo renal (in vivo stop-flow anterógrado), incrementó la actividad de kalikreína renal en la primera fracción de 140 µl de la orina colectada luego de dos minutos de la oclusión de uréter (p<0,05 y p<0,01). Aumento en la secreción de bicarbonato fue detectado en las primeras fracciones de 140 µl de orina del grupo control, que recibió sólo el vehículo, comparadas con las del grupo experimental, en el cual fue administrada kalikreína glandular en dosis de 1 µg (p<0,05) y 3 µg (p<0,01). Luego de la inyección retrógrada de kalikreína a través del uréter hacia los túbulos colectores corticales (stop-flow retrógrado), la concentración de bicarbonato referida a polyfructosan en fracción de orina fue significativamente inferior que la correspondiente al riñón contralateral control (p<0,05). Se observó incremento en la actividad de kalikreína renal en la misma fracción respecto del control (p<0,05). La confirmación de la llegada de las soluciones al segmento correspondiente al túbulo colector cortical fue realizada mediante estudios histológicos de microscopía óptica y electrónica de trasmisión, administrando por vía ascendente peroxidasa y sulfuro de mercurio coloidal. La integridad de las células intercalares y principales correspondientes a túbulo colector cortical fue evidenciada mediante microscopía electrónica de barrido. Estos resultados confirman y extienden datos previos (J Physiol 1995, Renal Physiol Biochemistry 1994), mostrando que la kalikreína renal actúa en el transporte de bicarbonato en membrana luminal del túbulo colector cortical